SOIL CONDITIONER, BIOFERTILIZER AND BIOPROTECTOR FOR INOCULATING PLANTS

Abstract

A bacterial product may comprise strains Azospirillum oryzae AO.sub.512, Azospirillum lipoferum AL.sub.20, Pantoea dispersa PD.sub.2, Bacillus subtilis TU.sub.2, Lysinibacillus sphaericus 3S and Paenibacillus polymyxa SR.sub.17, in a total minimal concentration of 10.sup.8 CFU/g (Colony forming Units). These bacteria may be attached to an insoluble granular carrier formed of a mixture of natural clays and minerals. This formulation may preserve the viability of the microorganism for a long time while in storage, as well as after its application on the field. These microorganisms, as PGPR, may be capable of fixing atmospheric nitrogen, solubilize phosphates, potassium, as well as other minerals immobilized in the soil, which allows for a drastic reduction, and even for a complete elimination, of conventional fertilization methods. These bacteria are not harmful in any sense neither to human beings nor the environment.

Claims

1. A biological fertilizer, and plant growth stimulator comprising: a. a pure culture of strain AO.sub.512 of the Azospirillum oryzae b. a pure culture of strain AL.sub.20 of the Azospirillum lipoferum c. a pure culture of strain PD.sub.2 of the Pantoea dispersa d. a pure culture of strain TU.sub.2 of the Bacillus subtilis e. a pure culture of strain S3 of the Lysinibacillus sphaericus f. a pure culture of strain SR.sub.17 of the Paenibacillus polymyxa (All these strains have to be deposited in a Type Culture Collection recognized under Budapest Treaty to meet the requirements on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (in USA could be NRRL (Agriculture Research Service od USDA) or ATCC (American Type Culture Collection)

2. The biological fertilizer of claim 1, wherein said cultures AO.sub.512, AL.sub.20, PD.sub.2, TU.sub.2, S3 and SR.sub.17 are immobilized in an insoluble granular carrier.

3. The biological fertilizer of claim 2, wherein said insoluble granular carrier is selected from the group consisting of clays; Vermiculite, Perlite, Sepiolite, Zeolites, nitrogen; Bone Fish Meal, Blood Meal, Feather Meal, Seaweed extract; phosphorus, Soft Phosphate Rock, Phosphate Rock, Bone Fish Meal, Vermicompost, Hydroxyapatite, manure, compost and guano, potassium; Green Sand, Feldspar-K, Mica, Kelp Meal, sylvite and sylvanite

4. The biological fertilizer of claim 1, wherein said cultures are formed from Plant Growth Promoting Rhizobacteria (PGPR).

5. The biological fertilizer of claim 1, wherein said fertilizer behaves as a slow-release system for releasing cells and nutrients.

6. The biological fertilizer of claim 1, wherein said fertilizer behaves as a plant growth stimulator.

7. The biological fertilizer of claim 1, behaves as fixer of atmospheric nitrogen and solubilizer of phosphorus, potassium and other minerals immobilized on the soil.

8. The biological fertilizer of claim 1, wherein said fertilizer behaves as a plant growth stimulator and soil conditioner.

9. The biological fertilizer of claim 1 wherein said fertilizer is applied once in the short-cycle crops and once a year in perennial crops; and

10. The biological fertilizer of claim 1 wherein said fertilizer confers resistance against abiotic stress and protect crops from pests and disease

11. The biological fertilizer of claim 1 wherein said fertilizer has a high stability, both in storage and during field applications

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0042] FIG. 1 Process flowchart of a method for producing an active composition of the biological fertilizer disclosed in the present invention.

[0043] FIGS. 2A-2F show the results of fertilization treatments in plants, obtained with the biological fertilizer described in the present invention.

[0044] FIGS. 3A-3G show results obtained in different tests where the biological fertilizer described in this invention was shown to be highly effective in combating known debilitating plant diseases.

[0045] FIGS. 4A, 4B are micrographic images of colonization of root surfaces by inoculating bacteria embodied in the present invention.

[0046] FIG. 5 is a graph illustrating growth of lettuce.

DESCRIPTION OF THE SOME EMBODIMENTS

[0047] Some embodiments of the inventive subject matter will now be described with reference to the drawings. Identical elements in the various figures are identified with the same reference numerals.

[0048] The ability to fix atmospheric nitrogen, solubilize phosphorus and potassium was used to the isolation and selection of the strains, to which, it was used selective culture media for each element in particular Nitrogen (EA Rodriguez Caceres 1981), Potassium (K B Prajapati and H A Modi 2012) and Phosphate (Mehta, S. and C S Nautiyal 2001). Once isolates strains of these three groups, the best ones were chosen in each group according, to their ability to fix N2, solubilize PO.sub.4.sup.3− or K and then, a screening was made taking in account the ability to stimulate plant growth. (Fernandez-Martinez A. I. 1996) using the fresh weight of wheat seed roots in vitro as the test model. In order to obtain a microbial consortium capable of supplying the plant with the 3 most important microelements (NPK), mixtures of strains were made, guaranteeing that each consortium include N2 fixers, and solubilizers of PO4 and K. With these mixtures, a solid product was prepared, wetting a previously prepared mineral mixture with the fermentation broths and then drying at a temperature not higher than 110° F. BF2 mixture was the best option, so it was decided to continue with this consortium that is composed of the strains AO.sub.512, AL.sub.20, PD.sub.2, Tu.sub.2, 3S and SR.sub.17.

Description of Some Implementations of the Product

[0049] In some implementations, the solid carrier may include a mixture of different raw materials, which basically include sources of nitrogen, phosphate, potassium, organic matter, microelements and an ion exchanger, in sufficient quantities for the product to begin to act and allow the establishment of the cells in the root and guarantee their interaction with the plant, these solids are added in a mixer of solids, until forming a homogeneous mixture. which in the example have the following composition:

Example of Product Composition

[0050]

TABLE-US-00002 Soil conditioner biofertilizer and biopesticide 1000 g Fermentation broth strains 100 mL Ammonium sulfate (in solution) 17 g Dipotassium phosphate (in solution) 4.5 g Zinc nitrate tetrahydrate (in solution) 0.12 mg Copper (ii) sulfate pentahydrate (in solution) 0.024 mg Manganese sulfate monohydrate (in solution) 0.12 mg Iron (ii) sulfate heptahydrate (in solution) 0.024 g Bone fish meal 12.5 g Phosphate rock 12.5 g Black earth humate 30 g Green sand 20 g Zeolite 0.5-1.5 mm 900 g

[0051] A description of the method for obtaining the product according some implementations is explained below as an example:

[0052] In some implementations, it is a Biofertilizer, plant growth and rooting enhancer, soil conditioner and bioprotector, to be used in both organic and conventional agriculture. In some implementations, it includes (or consists of in some implementations) viable cells of Azospirillum oryzae strain AO.sub.512, which is capable of fixing atmospheric nitrogen and has a high capacity to produce plant growth-regulating phytohormones, siderophores as well as the capacity of growing in feldspar as the only source of potassium. Azospirillum lipoferum AL.sub.20, able to fix of atmospheric nitrogen, which has a high capacity to produce phytohormones that stimulate plant growth and rooting. Pantoea dispersa, PD.sub.2, which is salt-tolerant, has a high capacity to produce organic acids an solubilize phosphate, produces siderophores, and shows antagonist activity against of phytopathogenic nematodes and Lysinibacillus sphaericus strain 3S which is a nitrogen fixing bacteria too and has great efficiency in the production of organic acids for the solubilization of phosphates and other nutrients of the soil, as well as antagonist of plant microbial diseases. Bacillus subtilis strain Tu.sub.2 that presents a good activity as stimulator of the vegetal growth, antagonist of microbial plant diseases, having also a moderate activity as nematicide, and Paenibacillus polymyxa strain SR.sub.17 that is able to fix atmospheric nitrogen, produce phytohormones, solubilize phosphates and potassium and have antagonistic activity against phytopathogenic fungi and bacteria. These bacteria isolated by the authors of the rhizosphere of plants that were growing in stressed condition. The preparation protects plants from abiotic stress and phytopathogenic organisms.

[0053] These bacteria may be grown in media that are not the subject of the present disclosure, but which contains only natural components. These bacteria may be adsorbed into a solid carrier containing clays and other natural raw materials. The final product may also incorporate mineral salts to facilitate bacterial growth in the soil.

[0054] These microorganisms may have been obtained by combining different methods of isolation, in soils of diverse characteristics, and selected from more than 100 different isolates. They have also been characterized as PGPR. The latter may give them a high biofertilizing and plant growth promoting capacity, that makes it possible to reduce or even eliminate the use of conventional fertilization in different crops. This is a universal product that may work very well in all types of crops and in multiple different environments, such as open fields, nurseries, greenhouses, as well as in gardens and golf courses.

[0055] To formulate the product, the different solid components of the composition may be mixed, to homogeneity and impregnated by the different solutions and fermentation broths, then dried at a temperature up to 110° F. to a residual moisture up to 6-8%, as shown in FIG. 1. Shown in FIG. 1 are steps in preparing the biofertilizing product according to some implementations. The process begins with mixing the ingredients, including the six bacteria species with a solid carrier. The components of the solid carrier have been previously listed in the summary section of this disclosure. Once thoroughly mixed in step, the mixture or consortium is thoroughly dried in step, then packed as appropriate or desired in step and stored in step 60 until application or delivery, which is the end of the process.

[0056] In one example the product obtained was periodically checked for cell viability in Potato-Tomato Agar solid medium, proving that it maintains more than 90% viability up to two years of conservation at room temperature, not higher than 95° F.

Examples of Stimulation of Plant Growth

Example 1

[0057] To test the ability of the bioproduct to stimulate plant growth, a greenhouse test was carried out using 300-mL capacity pots, filled with a peat-based substrate plus a common organic fertilizer mixture to function as control specimen. The same substrate as in the control specimen was also used for the treatment specimen using the biofertilization, to which or 50 g/L of the biofertilizer was added. Lettuce seedlings were used, with 20 repetitions per treatment. The plants were irrigated daily with water, throughout the 18 days that the experiment lasted from the moment of the transplant. FIG. 2A, demonstrates this evaluation, which Lettuce seedlings representing a control group before evaluation shown on the lower shelf marked “CONT” and the seedbed treated with the biofertilization, namely, +50 g/L shown on the top shelf. Note that plants consistently demonstrate a more exuberant growth and advance development then the plants in the control group.

[0058] Furthermore, it can be observed from the biofertilized group that there is a remarkable stimulation of the fresh weight of the plant in the treatment with the product, which shows that there has been a remarkable acceleration of the vegetative development of the plant in a relatively short period of time, as shown in the photo of both treatments before being evaluated. Obviously, the inoculation with the product has caused a remarkable increase in the growth, in relation to the control that has not been inoculated. An analysis of variance and a mean significance test were carried out on the results of the test, finding significant differences between the treatments that are shown in FIG. 2B.

Example 2

[0059] Treatment 1—BIOFERTILIZER 50 g/L+Miracle-Gro Garden Soil All Purpose (feeds up to 3-M).

[0060] Treatment 2 (control)—Miracle-Gro Garden Soil All Purpose (feeds up to 3-M).

Seeds—Cucumber seeds: Organic Ferry-Morse Cucumber Straight Eight

[0061] Pots—Square 250 ml pots and corresponding trays.

[0062] Planting of Seeds and Germination—Pots were filled with the appropriate media according to treatment, placed on the trays and were sprayed with water using a shower nozzle until saturation. Cucumbers were seeded at the rate of 2 seeds per pot and lightly covered with vermiculite and moss particles. A mist/fog nozzle was used to water-in the seeds. Seeds were planted and placed into a climate-controlled growth chamber. While in the growth chamber, the seeds were watered daily using a mist bottle. Once the seeds began to emerge, they were placed in a Greenhouse. Emerging plants were thinned to one plant per pot, leaving the stronger plant in both the controlled group and biofertilized group.

Plant Maintenance

[0063] While at the greenhouse, plants were monitored daily for any routine maintenance procedures (watering, spacing of trays, rotations of plants, etc.).

Data Collection

[0064] Foliar color measurements were collected on 3 sub-replicate plants per tray. Measurements were taken 3 and 4 weeks after planting. The foliar measurements were only collected from newly matured leaves. A SPAD 502DL Plus Chlorophyll Meter was used to collect the foliar color measurements. Foliar weights were collected on 3 sub-replicate plants per tray, recorded 4 weeks after planting and was obtained by cutting the plants and then weighed using a calibrated balance. Temperature and humidity data was recorded for the germination and growth while confined to a greenhouse.

Statistical Analysis

[0065] Raw data were transferred from this trial to data analysis software. JMP 11® software system was used to determine the means, pooled t, and ANOVA statistical analysis of the data.

Results and Discussion

[0066] Summaries of foliar color tones are presented in Tables 1 and 2 summarize the foliar weights achieved by the control group and the group fortified with the biofertilizer.

TABLE-US-00003 TABLE 1 Three weeks after seeding-foliar tones (individual plants) Minimum Maximum Mean Treatment SPAD units SPAD units SPAD units* BIOFERTILIZER + 44.80 68.80 52.39A Miracle-Gro Garden Soil All Purpose Miracle Gro-Garden Soil All Purpose 36.30 42.90 40.40B *Treatment means with no letters in common are significantly different from each other (P < 0.05) Treatment 1 plants had greater/darker foliar color tone than Treatment 2 plants after 3 weeks with mean of 52.39 SPAD units and 40.40 SPAD units respectively; the difference is statistically significant (P = 0.0002, F = 23.9487). (Table 1)

[0067] As presented in FIG. 2C and Table 2, trays comprising the treatment 1 group had significantly greater/heavier foliar weights than trays comprising treatment 2 group. The difference in foliar weights was even more pronounced after four weeks of growth (see FIG. 2D), with a mean of 249.393 g and 182.620 g respectively (P=0.0015, F=656.0020). Based on the results of this study, BIOFERTILIZER provided statistically significant darker foliar color tones at 3 and 4 weeks after seeding. It also achieved statistically heavier foliar weights 4 weeks after seeding.

TABLE-US-00004 TABLE 2 Four weeks after seeding-foliar weights trays Minimum Maximum Mean Treatment (grams) (grams) (grams)* BIOFERTILIZER + 245.94 254.26 249.39A Miracle Gro Garden Soil All Purpose Miracle Gro Garden Soil All Purpose 175.62 186.29 182.62B *Treatment means with no letters in common are significantly different from each other (P < 0.05)

[0068] Placing these findings in a visual context in FIG. 2E, the treatment 1 group is represented by the tray on the right, while treatment 2 group is represented by the tray on the left. The only difference between the two treatment groups is the presence of the biofertilizer disclosed herein. All other features, such as seed, growing conditions, soil substrate and care were kept constant with these groups. Treatment group 1 has visibly achieved a better and healthier progress than the treatment group 1, the result that must be attributed to the presence of the product disclosed herein.

Example 3. Cultivation of Lettuce in Soil at Full Cycle

[0069] To check the effectiveness of the product as a fertilizer in a complete cycle of a crop, an assay was carried out in which we proceeded to cultivate until maturation plants of lettuce, in separate beds that contained as a substrate, surface layer of soil (topsoil), performing 2 different treatments. Treatment 1-Flowerbed FIG. 2F, row of trays on the right, only had the superficial layer of soil, subjected to a scheme of conventional chemical fertilization for this type of crop. Treatment 2-Flowerbed, row on the left in FIG. 2F, contained the same superficial layer of soil, in which 5 g of product was placed under the hole at the time of the transplant. No fertilization was added to this treatment during the entire trial period.

The plants were watered daily and cut 48 days after the transplant. The results are shown in FIG. 5.

[0070] FIG. 5 demonstrates that there are significant differences between the statistics mean of both treatments—even if this is small—despite of the fact that control was fertilized periodically and in the other treatment it was only used in the biofertilizer once, at the beginning of the trial. This result demonstrates the ability of this product to completely replace conventional chemical fertilization in some crops.

[0071] FIGS. 3A-3G demonstrate the petri dishes containing various antagonists or phytopathogenic microorganisms, otherwise known as root and plant parasitic infections and diseases. In each figure, the pathogenic microorganism occupies the center of the petri dish, which is also the area not treated with the biofertilizer, while the indicia 100 indicates areas of the petri dish where an amount of biofertilizer has been deposited. Areas where biofertilizer has been deposited, and in many cases, surrounded areas as well, have been cleared of the pathogen. In practical applications, the biofertilizer, actively combats an existing disease. Additionally, bacteria comprising the biofertilizer is released at a slow pace through dry substrate, thus continuing to then inoculate a plant from the vanquished disease and other diseases. Leading to a more thorough colonization of the pant root system by the friendly bacteria as shown in FIGS. 4A and 4B, including the bacteria comprising the biofertilizer, and better synthesis of atmospheric nitrogen, phosphates, potassium and other minerals naturally found in the soil.

[0072] Although some implementations have been described with a certain degree of particularity, it is to be understood that the present disclosure has been made only by way of illustration and that numerous changes in the details of construction and arrangement of parts may be resorted to without departing from the spirit and the scope of the invention.

Embodiments

[0073] This section sets forth some embodiments of the inventive subject matter. However, this is not an exhaustive list of embodiments of the inventive subject matter described in this disclosure.

Embodiment 1—A biological fertilizer and plant growth stimulator comprises: [0074] a. a pure culture of strain AO.sub.512 of the Azospirillum oryzae [0075] b. a pure culture of strain AL.sub.20 of the Azospirillum lipoferum [0076] c. a pure culture of strain PD.sub.2 of the Pantoea dispersa [0077] d. a pure culture of strain TU.sub.2 of the Bacillus subtilis [0078] e. a pure culture of strain S3 of the Lysinibacillus sphaericus [0079] f. a pure culture of strain SR.sub.17 of the Paenibacillus polymyxa
All these strains may be deposited in a Type Culture Collection recognized under Budapest Treaty to meet the requirements on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (in USA could be NRRL (Agriculture Research Service od USDA) or ATCC (American Type Culture Collection).
Embodiment 2—The biological fertilizer of Embodiment 1, wherein said cultures AO.sub.512, AL.sub.20, PD.sub.2, TU.sub.2, S3 and SR.sub.17 are immobilized in an insoluble granular carrier.
Embodiment 3—The biological fertilizer any one or more of Embodiments 1-2, wherein said insoluble granular carrier is selected from the group consisting of clays; Vermiculite, Perlite, Sepiolite, Zeolites, nitrogen; Bone Fish Meal, Blood Meal, Feather Meal, Seaweed extract; phosphorus, Soft Phosphate Rock, Phosphate Rock, Bone Fish Meal, Vermicompost, Hydroxyapatite, manure, compost and guano, potassium; Green Sand, Feldspar-K, Mica, Kelp Meal, sylvite and sylvanite
Embodiment 4—The biological fertilizer of any one or more of Embodiments 1-3, wherein said cultures are formed from Plant Growth Promoting Rhizobacteria (PGPR).
Embodiment 5—The biological fertilizer of any one or more of Embodiments 1-4, wherein said fertilizer behaves as a slow-release system for releasing cells and nutrients.
Embodiment 6—The biological fertilizer of any one or more of Embodiments 1-5, wherein said fertilizer behaves as a plant growth stimulator.
Embodiment 7—The biological fertilizer of any one or more of Embodiments 1-6, behaves as fixer of atmospheric nitrogen and solubilizer of phosphorus, potassium and other minerals immobilized on the soil.
Embodiment 8—The biological fertilizer of any one or more of Embodiments 1-7, wherein said fertilizer behaves as a plant growth stimulator and soil conditioner.
Embodiment 9—The biological fertilizer of any one or more of Embodiments 1-8 wherein said fertilizer is applied once in the short-cycle crops and once a year in perennial crops.
Embodiment 10—The biological fertilizer of any one or more of Embodiments 1-9 wherein said fertilizer confers resistance against abiotic stress and protect crops from pests and disease [0080] 2. The biological fertilizer of Embodiments 1-10 wherein said fertilizer has a high stability, both in storage and during field applications