Endophytes for organic nitrogen use for sustainable agriculture

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 nitrogen fertilizers are provided. Endophytic microbial strains serve as biocatalysts to solubilize organic (proteinaceous) nitrogen otherwise unavailable to plants for their nutritional needs. Thus defined, biocatalysts will serve to replace synthetic nitrogen 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 fertilizer by 50% thus accomplishing optimal yields in technically and cost-effective manner.

Claims

1. A method for synthetically and systemically reducing nitrogen fertilizer requirement in any plant growth medium by purified homologous or heterologous endophytic bacteria producing protease enzymes, 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, and alkaline protease enzyme to inoculum density of 108 to 1010 cfu/ml; 2) suspending the said inoculum in sterile phosphate buffer saline medium to a concentration of 108 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 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.

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. 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. The method of claim 1 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.

9. The method of claim 1 wherein said endophytic inoculum comprises the ability to induce in the agricultural plant production of protease enzyme.

10. The method of claim 1 wherein the endophytic bacteria is obtainable from interior or exterior of the agricultural plant.

11. The method of claim 1 wherein the endophytic bacteria is obtainable from a different cultivar, variety or crop as compared to the seed.

12. The method of claim 1 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.

13. The method of claim 1 wherein the purified endophytic bacteria comprises a plurality of seed bacterial endophyte entities.

14. The method of claim 1 wherein the purified endophytic bacteria is combined with a purified fungal population.

15. A method comprising using NRRL B 67826 that is heterologous to the seed for the purpose of reducing nitrogen 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 nitrogen fertilizer that includes the following steps: 1) growing NRRL B 67826 having the ability to produce acid protease enzyme, and alkaline protease enzyme to a specific inoculum density of 108 to 1010 cfu/ml; 2) suspending the said inoculum in sterile phosphate buffer saline medium to a concentration of 108 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 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 nitrogen 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.

16. 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 nitrogen fertilizer in conventional and organic agriculture.

17. The method of claim 10 wherein NRRL B 67826 when applied to the said plant seed or plant growth medium can reduce or eliminate leaching of the applied nitrogen fertilizer in conventional and organic agriculture.

18. The method of claim 10 wherein the inoculated plant, plant part or seed is introduced in a plant growth medium to increase yield with optimal growth.

19. The method of claim 10, in which the said strain NRRL B 67826 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 67826 per gram or per milliliter of inoculant composition.

20. The method of claim 10, in which the said strain NRRL B 67826 is applied to the said plant seed one year before the said plant seed is planted in a plant growth medium.

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.

EXAMPLE 1: ISOLATING ENDOPHYTIC BACTERIA FROM FRESH PLANT SAMPLES

(9) 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

(10) 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.

(11) 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.

(12) 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.

(13) TABLE-US-00001 TABLE 2 Concentration of Endophytic bacteria Tyrosine produced isolated from corn (μg/ml) as an (C, T), surgarcane estimation of acid (S) and sorghum (J) protease activity J3/1 159.4 S-7P 66 J2/2 (BMS-101, 126 NRRL 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

(14) TABLE-US-00002 TABLE 3 Concentration of Endophytic bacteria Tyrosine produced isolated from corn (μg/ml) as an (C, T), surgarcane estimation of alkaline (S) and 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

(15) 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.

(16) 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.

(17) TABLE-US-00003 TABLE 4 Inoculation efficacy of endophytic bacteria in corn and sorghum cfu/ml (calculated using Sample description 1000 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
Un-Inoculated Corn and Sorghum Seeds Showed Zero Inoculum Density in Roots and Shoots

EXAMPLE 4

(18) 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.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 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)..sub.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.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. Poultry manure was added at the rate of g/L.

(19) 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.

(20) 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