Sustainable conventional and organic agriculture with reduced N and P fertilizer use

Abstract

Endophytic microbial strains as biocatalysts isolated from fresh plant samples, their compositions, and methods of use thereof to enhance the growth and/or yield of a plant in the presence of reduced (synthetic or otherwise) nitrogen and phosphorus fertilizers are provided. The selected endophytic microbial strains serve as biocatalysts to solubilize organic (proteinaceous) nitrogen otherwise unavailable to plants for their nutritional needs. In addition, selected endophytic microbial strains serve as biocatalysts to solubilize mineral-P and mineralize organic-P otherwise unavailable to plants for their nutritional phosphate needs. Thus defined, biocatalysts will serve to replace or reduce the requirement of (synthetic or otherwise) nitrogen and phosphorus fertilizers. Also provided are materials and methods for inoculating plants with these biocatalysts at carefully selected inoculum densities to reliably reduce the amount of nitrogen and phosphorus fertilizers by 30-50% thus accomplishing optimal yields in a cost-effective manner.

Claims

1. A novel synthetic combination comprising purified bacterial populations in association with a seed of an agricultural plant, wherein the purified bacterial populations comprise a plant bacterial endophyte that is heterologous to the seed, having all the identifying characteristics of NRRL accession no. B-67826, and is capable of producing protease to solubilize soil organic nitrogen at all relevant agricultural soil pHs, combined with another purified bacterial population comprising a plant bacterial endophyte that is heterologous to the seed having all the identifying characteristics of NRRL B 67827, and capable of producing organic acids, acid phosphatase, and alkaline phosphatase enzymes to solubilize soil phosphorus at acid and alkaline soil pHs and wherein the plant bacterial endophytes of NRRL accession no.'s B-67826 and B 67827 are both present on the surface of a seed or inside the seed and in an amount effective to improve nitrogen and phosphorus fertilizer use efficiency to increase plant's physiological properties for optimal plant growth and yield in organic and conventional agriculture compared to a reference agricultural plant grown under the same conditions in the absence of any purified bacterial endophyte treatments.

2. The synthetic combination of claim 1 which is made by first growing endophytic bacteria having all the identifying characteristics of NRRL B 67826 and NRRL B 67827 each to a specific inoculum density of 10.sup.8 to 10.sup.10 cfu/ml then suspending in sterile PBS medium to a concentration of 10.sup.8 cfu/ml, then preparing the seeds by surface sterilizing with 95% ethanol for 2 min and 2.5% sodium hypochlorite for 20-30 min followed by washing seven times in sterile water, then soaking the aforementioned surface sterile seeds in the said endophytic inoculum and henceforth placed in a temperature controlled incubator at 25 degree C. for exactly 30 minutes, then washing the thus prepared inoculated seeds with 70% alcohol for 2 minutes and with 2% sodium hypochlorite followed by washing with sterile water 5 times.

3. The synthetic combination of claim 1 wherein one or more seeds are of corn, sorghum, wheat, rice, and other vegetable, fruit, flower or grass plant and wherein the endophytic bacteria have all the identifying characteristics of NRRL B 67826 and NRRL B 67827 in the said seeds is capable to grow within the tissue of the said plants including its roots or shoots to a concentration of 10{circumflex over ( )}8 to 10{circumflex over ( )}10 cfu/g without reducing germination or yield.

4. The synthetic combination of claim 1, wherein the combination is combined with one or more of an insecticide, fungicide, nematicide, and biostimulant applied either as a seed treatment or as a foliar or soil rhizospheric treatment.

5. The synthetic combination of claim 1 wherein the plant bacterial endophytes are obtainable from a different cultivar, variety or crop as compared to the seed.

6. The synthetic combination of claim 1 wherein the nitrogen fertilizer can be nitrogen fertilizers used in conventional agriculture that are granular, liquid, or powder or a combination of these including but not limited to urea, anhydrous ammonia, urea ammonium nitrate, and ammonium nitrate.

7. The synthetic combination of claim 1 wherein the nitrogen fertilizer can be nitrogen fertilizers used in organic agriculture that are granular, liquid, or powder or a combination of these including but not limited to manure based fertilizers, composted fertilizers, high N− bat guano, and single cell based proteins/.

8. A novel synthetic combination comprising purified bacterial populations in association with a seed of an agricultural plant, wherein the purified bacterial populations comprise a plant bacterial endophyte that is heterologous to the seed, having all the identifying characteristics of NRRL accession no. B-67826, and is capable of producing protease to solubilize soil organic nitrogen at all relevant agricultural soil pHs, combined with another purified bacterial population comprising a plant bacterial endophyte that is heterologous to the seed having all the identifying characteristics of NRRL B 67827, and capable of producing organic acids, acid phosphatase, and alkaline phosphatase enzymes to solubilize soil phosphorus at acid and alkaline soil pHs and wherein the plant bacterial endophytes of NRRL accession no.'s B-67826 and B 67827 are both present on the surface of a seed or inside the seed and in an amount effective to increase yield in organic and conventional agriculture compared to a reference agricultural plant grown under the same conditions with no reduction in nitrogen and phosphorus fertilizers.

9. A method for synthetically and systemically improving nitrogen and phosphorus fertilizer use efficiency in a plant growth medium by purified homologous or heterologous endophytic bacteria producing protease enzymes, organic acids, alkaline phosphatase enzyme, and acid phosphatase enzyme sufficient to satisfy crop needs for optimal yields in said plant growth medium by colonizing the plant root and shoot at sufficient inoculum density that consists essentially of the following steps: 1) growing an endophytic bacteria having the ability to produce acid protease enzyme, alkaline protease, organic acids, alkaline phosphatase enzyme to inoculum density of 10.sup.8 to 10.sup.10 cfu/ml; 2) suspending the said inoculum in sterile phosphate buffer saline medium to a concentration of 10.sup.8 cfu/ml thereby providing an endophytic inoculum; 3) preparing the corn, sorghum, wheat, rice, and other vegetable, fruit, flower or grass seeds or plant parts by surface sterilizing with 95% ethanol for 2 min and 2.5% sodium hypochlorite for 20-30 min followed by washing seven times in sterile water; 4) soaking the aforementioned surface sterile seeds or plant parts in the said endophytic inoculum of step 2; 5) henceforth placing in a temperature controlled incubator at 25 degree C. for exactly 30 minutes with or without gentle shaking at 40-80 rpm, then washing the thus prepared inoculated seeds or plant part with 70% alcohol for 2 minutes and with 2% sodium hypochlorite followed by washing with sterile water 5 times; 6) treating the said prepared seeds or plant parts of step 5 with other seed treatments and coatings; 7) drying the said seed of step 6 before planting; and 8) planting the said prepared seeds or the said plant part in a plant growth medium with less than the recommended nitrogen and phosphorus fertilizer amount wherein the nitrogen is applied as urea, anhydrous ammonia, urea ammonium nitrate, ammonium nitrate, manure based fertilizers, composted fertilizers, high N− bat guano, single cell based proteins or another form and phosphorus is applied as is applied as triple superphosphate, diammonium phosphate, rock phosphate, manure or another form.

10. The method of claim 9 wherein in step 1, two or a plurality of endophytic bacteria are grown that each have optimal ability to produce acid proteas and alkaline protease enzymes and optimal ability to produce organic acids, acid phosphatase enzyme and alkaline phosphatase enzymes.

11. The method of claim 9, wherein the inoculated plant, plant part or seed is introduced in a plant growth medium in an amount effective to increase the yield of plants grown in said plant growth medium.

12. The method of claim 9 in which said endophytic inoculum, is introduced into a plant growth medium as part of an inoculant composition comprising at least 1×10.sup.5 colony forming units of said endophytic inoculum per gram or per milliliter of inoculant composition.

13. The method of claim 9 in which said treated plant part comprises at least 1×10.sup.5 colony forming units of said endophytic inoculum per kilogram of plant part.

14. The method of claim 9 in which said endophytic inoculum is introduced into a plant growth medium at a rate of at least 1×10.sup.5 colony forming units of said endophytic inoculum per acre of plant growth medium.

15. The method of claim 9 wherein said endophytic inoculum is applied to plant seeds, one year before said plant seed is planted in a plant growth medium.

16. The method of claim 9 wherein a formulation containing said endophytic inoculum comprises at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

17. The method of claim 9 wherein said endophytic inoculum comprises the ability to induce in the agricultural plant production of protease enzymes, organic acids, and phosphatase enzymes.

18. The method of claim 9 wherein the endophytic bacteria is obtainable from interior or exterior of the agricultural plant.

19. The method of claim 9 wherein the endophytic bacteria is obtainable from a different cultivar, variety or crop as compared to the seed.

20. The method of claim 9 wherein the inoculation of endophytic bacteria in step 4 comprises spraying, immersing, coating, encapsulating, injecting or dusting the seeds with a formulation containing said endophytic inoculum and at least one member selected from the group consisting of an agriculturally compatible carrier, a tackifier, a microbial stabilizer, a fungicide, an antibacterial agent, an herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIG. 1: A simplified view of the nitrogen cycle

(2) FIG. 2: Proportions of free amino acids (black bars), bound amino acids (dark grey bars), ammonium (light grey bars) and nitrate (white bars) in soil solutions adapted from Jamtgard et al., (2010).

(3) FIG. 3: Average nitrogen analysis of types of manure

(4) FIG. 4: Endophytic bacteria isolated from plants corn, sorghum, sugarcane: a) corn plants washed in tap water; b) separated roots and shoots; c) separated and chopped corn shoots for further surface sterilization and grinding to isolate endophytic bacteria

(5) FIG. 5: Endophytic bacteria isolated from corn plants

(6) FIG. 6: Endophytic bacteria isolated from sorghum plants

(7) FIG. 7: Endophytic bacteria isolated from sugarcane plants

(8) FIG. 8: a) control plants in N-deficient Hoagland solution; b) non-inoculated corn plant grown in complete Hoagland solution amended with 1 g poultry manure; c) non-inoculated corn plants grown in complete Hoagland solution; d) corn plant inoculated with J2/2 grown in N-deficient Hoagland solution amended with 1 g poultry manure.

(9) FIG. 9: a), b), and c) showing endophytic bacteria isolated from corn plants that solubilize based on sperber's PO.sub.4 solubilizing assay

(10) FIG. 10: a), b), c) and d) showing endophytic bacteria isolated from sorghum plants that solubilize based on sperber's PO.sub.4 solubilizing assay

(11) FIG. 11: a), b), c) d) and e) showing endophytic bacteria isolated from sugarcane plants that solubilize P.sub.i based on sperber's PO.sub.4 solubilizing assay

(12) FIG. 12: a) control plants in complete Hoagland solution; b) corn plant inoculated with T6 (BMS-201) grown in modified Hoagland solution amended with 0.2 g rock phosphate; c) corn plant inoculated with T6 (BMS-201) grown in modified Hoagland solution amended with 0.1 g iron-phosphate; d) and e) non-inoculated corn growing in phosphate deficient Hoagland solution

(13) FIG. 13: root and shoot length plotted for 1) control plant grown in phosphate deficient Hoagland solution show very poor growth as measured by their poor root and shoot lengths indicative of yield; 2) corn plant grown in complete Hoagland solution as expected are healthy and show good/optimal root and shoot growth; 3) corn plant grown in phosphate deficient Hoagland solution amended in iron-phosphate nanoparticles show root length and shoot lengths better than the reference/control plants; 4) corn plants grown in phosphate deficient Hoagland solution amended in rock-phosphate also show root length and shoot lengths better than the reference/control plants

(14) FIG. 14: Total chlorophyll content comparing T6, and T4 inoculated corn grown on rock phosphate (T4RP, T6RP) and iron phosphate, respectively (T4IP, T6IP), and control plants (D) and corn plants grown in full strength Hoagland solution (C)

(15) FIG. 15: a) sorghum control plants in complete Hoagland solution; b) sorghum plant inoculated with T6 grown in modified Hoagland solution amended with 0.1 g iron phosphate nanoparticles; c) sorghum plant inoculated with T6 grown in modified Hoagland solution amended with 0.2 g rock-phosphate; d) non-inoculated sorghum growing in phosphate deficient Hoagland solution

(16) FIG. 16: root and shoot length plotted for 1) control plant grown in phosphate deficient Hoagland solution show very poor growth as measured by their poor root and shoot lengths indicative of yield; 2) sorghum plant grown in complete Hoagland solution as expected are healthy and show good/optimal root and shoot growth; 3) T6 (BMS-201) inoculated sorghum plant grown in phosphate deficient Hoagland solution amended with iron-phosphate nanoparticles show root length and shoot lengths same as reference/control plants; 4) T6 (BMS-201) inoculated sorghum plants grown in phosphate deficient Hoagland solution amended with rock-phosphate show root length and shoot lengths slightly better than the reference/control plants.

(17) FIG. 17: A comparison of untreated Seed corn yields with combined T6 (BMS-201) and J2/2 (BMS-101) treated seed corn yields with no reduction in fertilizer. Combined T6 (BMS-201) and J2/2 (BMS-101) seed treatment yielded an 8.5 bushel/acre advantage compared to the untreated control.

(18) FIG. 18: A comparison of untreated spinach yields at 100% of recommended N and P fertilizers with spinach yields in combined T6 (BMS-201) and J2/2 (BMS-101) treatment with 50% reduction in both N and P fertilizers compared to recommended amount. T6 (BMS-201) and J2/2 (BMS-101) treated spinach showed excellent yields similar to the untreated spinach even with 50% reduction in N& P fertilizer amounts

EXAMPLE 1: ISOLATING ENDOPHYTIC BACTERIA FROM FRESH PLANT SAMPLES

(19) Fresh samples of corn, sorghum, and sugarcane plants were acquired and washed in tap water. The roots and shoots from each plant was separated. The roots and shoots were separated and chopped. They were then surface sterilized to eliminate any epiphytic bacteria and to facilitate isolation of only endophytic bacteria. The samples were then ground to isolate endophytic bacteria. Endophytic bacteria isolated from corn plants included T4, T6, and C8. Endophytic bacteria isolated from sorghum plants included J-1, J-2/1, J-2/2 (BMS-101, NRRL accession #B-67826), J-3/1, J-3/2, J-3/3, and J-4. Endophytic bacteria isolated from sugarcane plants included S-1/1, S-1/2, S-5, S-7, and S-8.

EXAMPLE 2: CHARACTERIZING ACID AND ALKALINE PROTEASE PRODUCTION FROM THE ISOLATED ENDOPHYTIC BACTERIA

(20) Acid protease assay: Protease activity was determined using the modified Anson method as described in Hashimoto et al., (1973). Briefly, reaction mixture was prepared by combining 1 ml of enzyme solution and 5 ml of bovine casein in 0.05 M acetate buffer (pH 2.5) and incubated at 30 degree C. for 10 minutes. Five ml of 0.11 M trichloroacetic acid was added to stop the reaction. The reaction mixtures was filtered after incubation at 30 degree C. for 30 minutes. Five ml of 0.55 M sodium carbonate was added to 2 ml of the filtrate followed by the addition of 1 ml of thrice diluted phenol reagent. Acid protease activity exuded by endophytic bacteria was quantified based on the concentration of tyrosine measured at 660 nm.

(21) Alkaline protease assay: Alkaline protease activity was determined according to the procedure of Smita et al., (2012). The assay medium (50 ml) containing nutrient broth and 1% casein at pH 10 was inoculated with each isolate and incubated at 37 degree C. for 48 hrs in a water bath shaker. After incubation, the broth cultures were centrifuged at 10,000 rpm for 10 minutes at 4 degree C. and the supernatant was collected to determine the activity of alkaline protease. The activity of alkaline protease was determined similar to the procedure described above for acid protease by measuring the release of tyrosine at 280 nm. The main difference was that the pH was adjusted to pH 10 instead of 2.5 by addition of Glycine-NaOH buffer.

(22) Because Acid-protease are very effective at the common soil pHs we selected J2/2 (BMS-101, NRRL accession #B-67826) an endophytic bacteria isolated from sorghum and tested its inoculation efficacy in corn and sorghum.

(23) TABLE-US-00001 TABLE 2 Endophytic bacteria Concentration of isolated from Tyrosine produced corn (C, T), (μg/ml) as an surgarcane (S) estimation of acid and sorghum (J) protease activity J3/1 159.4 S-7P 66 J2/2 (BMS-101, NRRL 126 accession # B-67826) S-1/2 55.7 J-4 112.6 T-4 24.8 T-6 18.5 S-8 46.4 S-7 54 S-5 58.4 S-1/12 42.8 J2/1 102.6 J-3/3 52.6 J-1 94 C-8 44.9

(24) TABLE-US-00002 TABLE 3 Concentration Endophytic of Tyrosine bacteria isolated produced (μg/ml) from corn (C, T), as an estimation surgarcane (S) and of alkaline sorghum (J) protease activity S-1/2 77 S-8 89 S-7 P 97 S-1/3 87

EXAMPLE 3: TESTING INOCULATION EFFICACY OF ENDOPHYTIC BACTERIA

(25) The inoculum for endophytic bacteria was grown under controlled conditions for 48 hrs to inoculum density of 10.sub.8 to 10.sub.10 cfu/ml. The inoculum was centrifuged and suspended in sterile PBS to a concentration of 10.sub.8 cfu/ml. The seeds were surface sterilized with 95% ethanol for 2 min and 2.5% sodium hypochlorite for 20-30 min followed by washing seven times in sterile water. Surface sterilized seeds were soaked in sterile PBS containing endophytic bacteria and placed in a temperature controlled incubator shaker at 25 degree C. for exactly 30 minutes. The inoculated seeds were washed with 70% alcohol for 2 minutes and with 2% sodium hypochlorite followed by washing with sterile water 5 times. The surface sterilized seeds were placed in sterile petriplates containing 0.7% of water agar, 5-10 seeds per plate. The seed containing plates were transferred to growth chamber set at 30 degree C. and left for 48 hours to germinate. Well germinated seeds with shoot and roots were separated and surface sterilize with 95% of ethanol for 5 min and 20 min with 4% sodium hypochlorite followed by 4-5 times sterile water rinse. The water rinsed root and shoot parts were transferred to PBS containing solution and ground to rapture the tissue. 1 ml of ground tissue was diluted in 9 ml of sterile water serial dilutions were continued to obtain 100 and 1000 fold dilution and spread on nutrient agar plates. After growth the colonies were counted and tabulated. Non-inoculated seeds served as negative controls.

(26) Based on the amounts of protease production J3/1 and J2/2 (BMS-101, NRRL accession #B-67826) were selected for testing their inoculation efficacy in corn and sorghum. Because J3/1 inoculum density measurements in roots and shoot replicates showed wide variability, J2/2 (BMS-101, NRRL accession #B-67826) was chosen as the protease producing strain for further experiments. The non-inoculated control seeds showed zero inoculum density in the root and shoots.

(27) TABLE-US-00003 TABLE 4 Inoculation efficacy of endophytic bacteria in corn and sorghum cfu/ml (calculated using 1000 Sample description fold dilution) J2/2 (BMS-101, NRRL accession # B-67826) 2.4 × 10.sup.6 inoculum density in corn root J2/2 (BMS-101, NRRL accession # B-67826) 3.1 × 10.sup.5 inoculum density in corn shoot J2/2 (BMS-101, NRRL accession # B-67826) 6.6 × 10.sup.6 inoculum density in sorghum root J2/2 (BMS-101, NRRL accession # B-67826) 1.8 × 10.sup.6 inoculum density in sorghum shoot Un-inoculated corn and sorghum seeds showed zero inoculum density in roots and shoots

EXAMPLE 4

(28) Standard Hoagland solutions (hydroponic nutrient solutions) were prepared according to the composition in (ref) and contained Ca(NO.sub.3).sub.2.4H.sub.2O, NH.sub.4NO.sub.3, KCl, KNO.sub.3, Mg(NO.sub.3).sub.2.6H.sub.2O, KH.sub.2PO.sub.4, Fe(NO.sub.3).sub.3.9H.sub.20, Na HEDTA, MnCl.sub.2.4H.sub.20, H.sub.3BO.sub.3, ZnSO.sub.4.7H.sub.2O, CuSO.sub.4.5H.sub.2O, and Na.sub.2MoO.sub.4.2H.sub.20. The young corn seedlings cannot tolerate full strength Hoagland solution. Hence 1/2 strength Hoagland solution was used from VE to V1 vegetative stage. The plants were grown until V3 vegetative growth stage because nitrogen deficiency symptoms can be observed during V1 to V3 growth stage. Nitrogen deficient Hoagland solution was prepared by eliminating all sources of nitrates and ammonium nitrogen and contained CaCl.sub.2.2H.sub.2O, KCl, K.sub.2SO.sub.4, MgCl.sub.2.6H.sub.2O, KH.sub.2PO.sub.4, FeCl.sub.3.6H.sub.2O, Na HEDTA, MnCl.sub.2.4H.sub.20, H.sub.3BO.sub.3, ZnSO.sub.4.7H.sub.2O, CuSO.sub.4.5H.sub.2O, and Na.sub.2MoO.sub.4.2H.sub.20. Poultry manure was added at the rate of g/L.

(29) The viable inoculated seeds were placed in a muslin cloth and washed with running tap water and dried by placing on autoclaved tissue paper. The seeds were surface sterilized in laminar flow hood with 70% alcohol for 30 minutes and then by washing with 10% sodium hypochlorite solution for 20 minutes. After surface sterilization, the seeds were washed with sterile water, excess water removed by blotting with autoclaved tissue paper, and then air dried under laminar flow. Five to ten surface sterilized and dried seeds were then placed in sterile petriplates containing water soaked blotting paper and transferred to growth chamber maintained at 25-27 degree C. and left for 48 hours to germinate. After seeds germinated, they were transferred to hydroponic reactors. Hydroponic reactors constituted of root permeable plastic buckets. Air pump with air controllers were used to provide aeration to the plants in hydroponic systems and all systems were maintained under greenhouse conditions at a temperature of 24±2 degree C. and relative humidity of 70±3%. After control plants showed nitrogen deficiency symptoms, the plants were removed from hydroponic reactors and washed under running tap water to completely remove Hoagland solution. The plant was dried with blotting paper while taking care to not damage the roots.

(30) Reduced leaf area and degradation of chlorophyll in leaves is symptomatic of nitrogen deficiency so we measured chlorophyll a and chlorophyll b in plants. A single leaf per plant was used for obtaining 10 leaf discs of 1 cm each and weighed. Five of these leaf discs were placed per tube containing 5 ml of 1:1 ratio of DMSO:acetone and the tubes were placed in the dark overnight to allow chlorophyll leaching. After chlorophyll leaching the solution turns green and the concentration of chlorophyll in leaves is calculated by measuring absorbance at 645, and 663 nm. The total chlorophyll is estimated using the following formulae:
Chlorophyll II a (g/l)=0.0127 A.sub.663−0.00269 A.sub.645
Chlorophyll II b (g/l)=0.0029 A.sub.663−0.00468 A.sub.645
Total Chlorophyll (g/l)=0.0202 A.sub.663+0.00802 A.sub.645

EXAMPLE 5: TESTING ENDOPHYTIC BACTERIA FOR INORGANIC-P SOLUBILIZATION

(31) Sperber's media for screening Pi solubilizing endophytic bacteria: The basal Sperber (1958) medium was used and contained glucose 10.0 g/l, yeast extract 0.5 g/l, CaCl.sub.2 0.1 g/l, MgSO.sub.4.7H.sub.2O 0.25 g/l and agar 15.0 g/l. The medium was supplemented with 2.5 g/L of Ca.sub.3(PO.sub.4).sub.2 (TCP-tricalcium phosphate) as P source to appraise the ability of the strains to mobilize inorganic P sources. The pH of the medium was adjusted to 7.2 before autoclaving. The media were distributed in 9 cm diameter Petri plates and marked in four equal parts after solidification. Using the drop plate method, each part was inoculated with innocula. All tests were performed with four replications. Inoculated plates were incubated in dark at 27 degree C. and the diameter of clear zone (halo) surrounding the bacterial growth as well as the diameter of colony were measured after 10, 20 and 30 days.

(32) TABLE-US-00004 TABLE 1 Results of Sperber's PO.sub.4 solubilizing Assay Endophytic bacteria Phosphate isolated from corn solubilizing (C, T), surgarcane activity (S) and sorghum (J) (zone in cm) C-8 Very low (0.1 cm) T-4 Medium (1 cm) T-6 Medium (0.6 cm) J-2/2 Very low (0.2 cm) J-3/2 Very low (0.1 cm) J-3/1 Very low (0.3 cm) J-4 Medium (1 cm) S-1/1 Very low (0.1 cm) S-1/2 Very low (0.3 cm) S-5 Very high (4 cm) S-7 Very high (5 cm) S-8 High (3 cm)

EXAMPLE 6: TESTING ENDOPHYTIC BACTERIA FOR ACID PHOSPHATASE PRODUCTION

(33) Screening for acid phosphatase producing endophytic bacteria: The isolated strains were grown in 50 ml of liquid medium (0.1% Ca-phytate; 1.5% glucose; 0.2% NH.sub.4NO.sub.3; 0.05% KCl; 0.05% MgSO.sub.4.7H.sub.2O; 0.03% MnSO.sub.4.4H.sub.2O; 0.03% FeSO.sub.4.7H.sub.2O, pH 5.5) in 500-ml flask and incubated at 28 degree C. for 48 hours on reciprocal shaker (200 rpm). The cells were collected from 1 ml of culture by centrifugation at 5000×g for 10 minutes in cool room (40 C) and re-suspended in acetate buffer (0.2 M, pH 5.5). The reaction mixture was prepared. It consisted of 0.8 ml acetate buffer (0.2 M, pH 5.5) containing I mM Na-phytate and 0.2 ml of cell suspension. After incubation for 30 minutes at 37 degree C., the reaction was stopped by adding 1 ml of trichloroacetic acid. One ml aliquot was analyzed for inorganic phosphate liberated using the colorimetric procedure. One unit of enzyme activity was defined as the amount of enzyme liberating 1 n mol of inorganic phosphate per minute.

(34) TABLE-US-00005 TABLE 2 Results of acid phosphatase assay Endophytic bacteria Concentration of isolated from corn acid phosphatase (C, T), surgarcane produced (S) and sorghum (J) (mg/l) J-1 0.60 S-5 0.60 C-8 0.58 J-4 0.57 S-7 0.62 J-2/2 0.52 T6 0.60 J3/1 0.62 T4 0.61 S-1/2 0.60 J-2/1 0.59

EXAMPLE 7: TESTING ENDOPHYTIC BACTERIA FOR ALKALINE PHOSPHATASE PRODUCTION

(35) Screening for alkaline phosphatase producing endophytic bacteria: The endophytic bacteria were grown in blood agar for 24 h. One colony was transferred and incubated at 37 degree C. in 2.75 ml of propanediol buffer (0.2 mol/liter, pH 7.5) containing 2 mg of 5-bromo-4-chloro-3-indolyl phosphate previously dissolved in 0.25 ml of N,N-dimethyl formamide. 0.2 ml of MgCl.sub.2 (5 mmol/liter) was added as an activator. Alkaline phosphatase production was examined every 30 minutes for 4 h by looking for a blue-green indigo precipitate development on the bacterial growth causing the entire solution to become blue.

(36) TABLE-US-00006 TABLE 3 Results of alkaline phosphatase assay Endophytic bacteria isolated from corn (C, T), surgarcane Change of color to (S) and sorghum (J) indigo blue-green C-8 Negative T-4 Negative T-6 Positive J-1 Negative J-2/1 Negative J-2/2 Negative J-3/2 Negative J-3/1 Negative J-3/3 Negative J-4 Negative S-1/1 Negative S-1/2 Negative S-5 Negative S-7 Negative S-8 Negative

EXAMPLE 8: TESTING INOCULATION EFFICACY OF ENDOPHYTIC BACTERIA

(37) The inoculum for endophytic bacteria was grown under controlled conditions for 48 hrs to inoculum density of 10.sup.8 to 10.sup.10 cfu/ml. The inoculum was centrifuged and suspended in sterile PBS to a concentration of 10.sup.8 cfu/ml. The seeds were surface sterilized with 95% ethanol for 2 min and 2.5% sodium hypochlorite for 20-30 min followed by washing seven times in sterile water. Surface sterilized seeds were soaked in sterile PBS containing endophytic bacteria and placed in a temperature controlled incubator shaker at 25 degree C. for exactly 30 minutes. The inoculated seeds were washed with 70% alcohol for 2 minutes and with 2% sodium hypochlorite followed by washing with sterile water 5 times. The surface sterilized seeds were placed in sterile petriplates containing 0.7% of water agar, 5-10 seeds per plate. The seed containing plates were transferred to growth chamber set at 30 degree C. and left for 48 hours to germinate. Well germinated seeds with shoot and roots were separated and surface sterilize with 95% of ethanol for 5 min and 20 min with 4% sodium hypochlorite followed by 4-5 times sterile water rinse. The water rinsed root and shoot parts were transferred to PBS containing solution and ground to rapture the tissue. 1 ml of ground tissue was diluted in 9 ml of sterile water serial dilutions were continued to obtain 100 and 1000 fold dilution and spread on nutrient agar plates. After growth the colonies were counted and tabulated. Non-inoculated seeds served as negative controls.

(38) TABLE-US-00007 TABLE 4 Inoculation efficacy of endophytic bacteria in corn and sorghum cfu/ml (calculated using 1000 Sample description fold dilution) Corn-root inoculated with T6 (BMS-201; 2.1 × 10.sup.6 NRRL accession number B-67827) Corn-shoot inoculated with T6 (BMS-201; 1.7 × 10.sup.5 NRRL accession number B-67827) Sorghum-root inoculated with T6 (BMS-201; 2.6 × 10.sup.6 NRRL accession number B-67827) Sorghum-shoot inoculated with T6 (BMS-201; 3.4 × 10.sup.5 NRRL accession number B-67827) Un-inoculated corn and sorghum seeds showed zero inoculum density in roots and shoots

EXAMPLE 9: TESTING THE EFFICACY OF ENDOPHYTIC INOCULATION FOR ELIMINATING CHEMICAL FERTILIZERS IN CONTROLLED HYDROPONIC SYSTEMS

(39) Standard Hoagland solutions (hydroponic nutrient solutions) were prepared and contained Ca(NO.sub.3).sub.2.4H.sub.2O, NH.sub.4NO.sub.3, KCl, KNO.sub.3, Mg(NO.sub.3).sub.2.6H.sub.2O, KH.sub.2PO.sub.4, Fe(NO.sub.3).sub.3.9H.sub.20, Na HEDTA, MnCl.sub.2.4H.sub.20, H.sub.3BO.sub.3, ZnSO.sub.4.7H.sub.2O, CuSO.sub.4.5H.sub.2O, and Na.sub.2MoO.sub.4.2H.sub.20. The young corn seedlings cannot tolerate full strength Hoagland solution. Hence 1/2 strength Hoagland solution was used from VE to V1 vegetative stage. The plants were grown until V3 vegetative growth stage because phosphate deficiency symptoms can be observed during V1 to V3 growth stage. Phosphate deficient Hoagland solution was prepared by eliminating KH.sub.2PO.sub.4.

(40) The viable inoculated seeds were placed in a muslin cloth and washed with running tap water and dried by placing on autoclaved tissue paper. The seeds were surface sterilized in laminar flow hood with 70% alcohol for 30 minutes and then by washing with 10% sodium hypochlorite solution for 20 minutes. After surface sterilization, the seeds were washed with sterile water, excess water removed by blotting with autoclaved tissue paper, and then air dried under laminar flow. Five to ten surface sterilized and dried seeds were then placed in sterile petriplates containing water soaked blotting paper and transferred to growth chamber maintained at 25-27 degree C. and left for 48 hours to germinate. After seeds germinated, they were transferred to hydroponic reactors. Hydroponic systems were maintained under greenhouse conditions at 23-24 degree C., 70% relative humidity, and 12 hours photo-period. Seeds were first grown in full strength Hoagland solution and then transplanted into hydroponic reactors for all the treatments and controls. Five replications were used for all treatments and controls.

(41) Hydroponic reactors constituted of root permeable plastic buckets. Air pump with air controllers were used to provide aeration to the plants in hydroponic systems and all systems were maintained under greenhouse conditions at a temperature of 24±2 degree C. and relative humidity of 70±3%. After control plants showed phosphate deficiency symptoms, the plants were removed from hydroponic reactors and washed under running tap water to completely remove Hoagland solution. The plant was dried with blotting paper while taking care to not damage the roots. The roots were separated from the shoots by using sharp scissors and were weighed to record fresh weight of samples. The root and shoot samples were also dried in the dry air oven at 40 degree C. for 2 days and their dry weight was recorded. The root:shoot ratio of individual plants was calculated and recorded.

(42) Reduced leaf area and degradation of chlorophyll in leaves is also symptomatic of phosphate deficiency so we also measure chlorophyll a and chlorophyll b in plants. A single leaf per plant was used for obtaining 10 leaf discs of 1 cm each and weighed. Five of these leaf discs were placed per tube containing 5 ml of 1:1 ratio of DMSO:acetone and the tubes were placed in the dark overnight to allow chlorophyll leaching. After chlorophyll leaching the solution turns green and the concentration of chlorophyll in leaves is calculated by measuring absorbance at 645, and 663 nm. The total chlorophyll is estimated using the following formulas:
Chlorophyll II a (g/l)=0.0127 A.sub.663−0.00269 A.sub.645
Chlorophyll II b (g/l)=0.0029 A.sub.663−0.00468 A.sub.645
Total Chlorophyll (g/l)=0.0202 A.sub.663+0.00802 A.sub.645