Phosphorus fertilizer bio-catalyst for sustainable crop production

Abstract

Endophytic microbial strains as biocatalysts isolated from fresh plant samples, compositions, and methods of use thereof to enhance the growth and/or yield of a plant in the presence of reduced synthetic phosphate fertilizers are provided. 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 synthetic phosphate fertilizers. Also provided are materials and methods for inoculating plants with these biocatalysts at carefully selected inoculum densities to reliably reduce the amount of synthetic phosphate fertilizer by 50% thus accomplishing obtaining optimal yields in technically and cost-effective manner.

Claims

1. A method comprising using a plant bacterial endophyte that is heterologous to the seed for the purpose of reducing phosphorus fertilizer requirement for optimal plant growth and yield in organic and conventional agriculture compared to a reference agricultural plant grown under the same conditions with no reduction in phosphorus fertilizer that consists essentially of the following steps: 1) growing an endophytic bacteria having the ability to produce organic acids, acid phosphatase enzyme, and alkaline phosphatase enzyme to a 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 phosphorus fertilizer amount wherein the phosphorous is applied as triple superphosphate, diammonium phosphate, rock phosphate, manure or another form.

2. The method of claim 1, 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.

3. The method of claim 1, 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.

4. The method of claim 1, in which said endophytic inoculum, is introduced into a plant growth medium as part of a treated plant part.

5. The method of claim 4, 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.

6. The method of claim 1, 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.

7. A The method of claim 1 wherein said endophytic inoculum is applied to plant seeds, one year before said plant seed is planted in a plant growth medium.

8. A method comprising using NRRL B 67827 that is heterologous to the seed for the purpose of reducing phosphorus fertilizer requirement for optimal plant growth and yield in organic and conventional agriculture compared to a reference agricultural plant grown under the same conditions with no reduction in phosphorus fertilizer that includes the following steps: 1) growing NRRL B 67827 having the ability to produce organic acids, acid phosphatase enzyme, and alkaline phosphatase enzyme to a specific 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 vegetable, fruit, flower, or grass seed, 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 seed or plant parts in the said endophytic inoculum; and 5) henceforth placing in a temperature controlled incubator at 25 degree C. or any other temperature dependent on seed type or plant parts with or without gentle shaking at 40-80 rpm for exactly 30 minutes, then washing the thus prepared inoculated seeds or plant parts 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 phosphorus fertilizer amount wherein the phosphorous is applied as triple superphosphate, diammonium phosphate, rock phosphate, manure or another form.

9. The method of claim 1 in which said endophytic inoculum when applied to the said plant seed or plant growth medium can reduce or eliminate leaching of the applied phosphorus fertilizer in conventional and organic agriculture.

10. The method of claim 8 wherein NRRL B 67827 when applied to the said plant seed or plant growth medium can reduce or eliminate leaching of the applied phosphorus fertilizer in conventional and organic agriculture.

11. The method of claim 8 wherein the inoculated plant, plant part or seed is introduced in a plant growth medium to increase yield with optimal growth.

12. The method of claim 8, in which the said strain NRRL B 67827 is introduced into a plant growth medium as part of an inoculant composition comprising at least 1×10.sup.5 colony forming units of NRRL B 67827 per gram or per milliliter of inoculant composition.

13. The method of claim 8, in which the said strain NRRL B 67827 is applied to the said plant seed one year before the said plant seed is planted in a plant growth medium.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1: 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

(2) FIG. 2: Endophytic bacteria isolated from corn plants

(3) FIG. 3: Endophytic bacteria isolated from sorghum plants

(4) FIG. 4: Endophytic bacteria isolated from sugarcane plants

(5) FIG. 5: a), b), and c) showing endophytic bacteria isolated from corn plants that solubilize P; based on sperber's PO.sub.4 solubilizing assay

(6) FIG. 6: a), b), and c) showing endophytic bacteria isolated from sorghum plants that solubilize P; based on sperber's PO.sub.4 solubilizing assay

(7) FIG. 7: a), b), and c) showing endophytic bacteria isolated from sugarcane plants that solubilize P.sub.i based on sperber's PO.sub.4 solubilizing assay

(8) FIG. 8: a) control plants in complete Hoagland solution; b) corn plant inoculated with T6 grown in modified Hoagland solution amended with 0.2 g rock phosphate; c) corn plant inoculated with T6 grown in modified Hoagland solution amended with 0.1 g iron-phosphate; d) and e) non-inoculated corn growing in phosphate deficient Hoagland solution

(9) FIG. 9: 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

(10) FIG. 10: 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)

(11) FIG. 11: 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

(12) FIG. 12: 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) 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) 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.

EXAMPLE 1: ISOLATING ENDOPHYTIC BACTERIA FROM FRESH PLANT SAMPLES

(13) Fresh samples of corn, sorghum, and sugarcane plants were acquired and washed in tap water.

(14) The roots and shoots from each plant 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, 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: TESTING ENDOPHYTIC BACTERIA FOR INORGANIC-P SOLUBILIZATION

(15) 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.

(16) TABLE-US-00001 TABLE 1 Results of Sperber's PO.sub.4 solubilizing Assay Endophytic bacteria isolated from corn (C, T), surgarcane (S) and Phosphate solubilizing sorghum (J) activity (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 3: TESTING ENDOPHYTIC BACTERIA FOR ACID PHOSPHATASE PRODUCTION

(17) 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 1 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.

(18) TABLE-US-00002 TABLE 2 Results of acid phosphatase assay Endophytic bacteria isolated from corn (C, T), Concentration of acid surgarcane (S) and phosphatase produced 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 4: TESTING ENDOPHYTIC BACTERIA FOR ALKALINE PHOSPHATASE PRODUCTION

(19) 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.

(20) TABLE-US-00003 TABLE 3 Results of alkaline phosphatase assay Endophytic bacteria isolated from corn (C, T), surgarcane (S) and Change of color to 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 5: TESTING INOCULATION EFFICACY OF ENDOPHYTIC BACTERIA

(21) 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.

(22) TABLE-US-00004 TABLE 4 Inoculation efficacy of endophytic bacteria in corn and sorghum cfu/ml (calculated using Sample description 1000 fold dilution) Corn-root inoculated with T6 2.1 × 10.sup.6 Corn-shoot inoculated with T6 1.7 × 10.sup.5 Sorghum-root inoculated with T6 2.6 × 10.sup.6 Sorghum-shoot inoculated with T6 3.4 × 10.sup.5 Un-inoculated corn and sorghum seeds showed zero inoculum density in roots and shoots

EXAMPLE 6: TESTING THE EFFICACY OF ENDOPHYTIC INOCULATION FOR ELIMINATING CHEMICAL FERTILIZERS IN CONTROLLED HYDROPONIC SYSTEMS

(23) 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.2O, Na HEDTA, MnCl.sub.2.4H.sub.2O, 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.2O. The young corn seedlings cannot tolerate full strength Hoagland solution. Hence ½ 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.

(24) 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.

(25) 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.

(26) 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