Claims:1. A composition for increasing phosphate availability in soil, the composition comprising:
- an isolated strain of Bacillus siamensis having the deposit accession number MCC 0201; and
- fructooligosaccharide.
2. The composition as claimed in claim 1, wherein said Bacillus strain is coated with fructooligosaccharide.
3. The composition as claimed in claim 1 or 2, wherein the composition comprises of said Bacillus strain in an amount of 10^6 to 10^10 cfu per unit of the composition.
4. The composition as claimed in claim 1 or 2, wherein the composition comprises of fructooligosaccharide in an amount of 55-95 % w/w of the composition.
5. The composition as claimed in claim 1 or 2, further comprising a phosphorus source in an amount of 0.5 to 1 % w/w of the composition.
6. The composition as claimed in claim 1 or 2, further comprising a micronutrient in a concentration of about 10 to 90 % (by weight) of the composition, said micronutrient being selected from a group consisting of Zinc, Boron, Copper, Iron, Manganese, and Molybdenum.
7. The composition as claimed in claim 1 or 2, further comprising one or more of pesticides, acaricides, insecticides and/or nematicides.
, Description:Field of Invention
The present disclosure relates to a composition for increasing phosphate availability in soil.
Background
Phosphorus, next to nitrogen, is the most crucial nutrient for crop production. The availability of phosphorus in soil is influenced by soil organic matter, pH, and exchangeable and soluble forms of Al, Fe, and Ca. In acidic soils, phosphorus is deficient because soluble inorganic phosphorus is fixed by Al and Fe. In alkaline or pH-neutral soils, phosphorus reacts with calcium and becomes inaccessible. Thus, the total phosphorus content of arable soils varies from 0.02–0.5% with an average of 0.05% in both inorganic and organic forms. As a result, higher plants growth depends on diffusion processes and a continuous release from insoluble sources to meet their phosphorus demand.
Conventionally, large amounts of lime are used to saturate Al and Fe ions and increase availability of phosphorus. However, this approach has not been successful because it is not economical. The practice is also not environmentally friendly. For example, over-liming precipitates P ions with Ca as Calcium phosphate.
Also, inorganic P fertilizers such as phosphate rocks and Triple Superphosphate (TSP) have been applied to soil to increase phosphorus content in the soil. However, excessive use of P fertilizers causes eutrophication. Also, it is quickly converted in soil into relatively unavailable forms.
Recently, attempts have been made to use biofertilizers to provide nutrients for plant growth. The biofertilizers use different types of microorganisms to improve the availability of nutrients in soil. Biofertilizers are advantageous as they are environment friendly. Microrganisms have been used to improve availability of phosphorus in soil. For example, species of Bacillus, Pseudomonas, Rhizobium, Aspergillus and Penicillium have been used for improving phosphorus availability in soil.
Summary
A composition for increasing phosphate availability in soil is disclosed. Said composition comprises an isolated strain of Bacillus siamensis having the deposit accession number MCC 0201, and fructooligosaccharide.
Detailed Description
For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alterations and further modifications in the disclosed composition and method, and such further applications of the principles of the disclosure therein being contemplated as would normally occur to one skilled in the art to which the disclosure relates.
It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the disclosure and are not intended to be restrictive thereof.
Reference throughout this specification to “one embodiment” “an embodiment” or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, appearances of the phrase “in one embodiment”, “in an embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.
As used herein, the term "isolate, isolates, isolating, and/or isolated, etc." is intended to mean that the referenced material is removed from the environment in which it is normally found.
As used herein, terms "phosphate solubilization", or "phosphate solubilizing", etc. are intended to mean the conversion of insoluble phosphate (e.g., rock phosphate, etc.) into a soluble phosphate form.
As used herein, term “unit” is intended to mean units of measurement- grams and milliliter.
In its broadest scope, the present disclosure relates to a composition for increasing phosphate availability in soil. Specifically, the disclosed composition comprises an isolated strain of Bacillus siamensis having the deposit accession number MCC 0201 and fructooligosaccharide.
It was found by present inventors that said Bacillus strain having the deposit accession number MCC 0201 produces higher amount of organic acids as compared to known strains of Bacillus, particularly, in the presence of fructooligosaccharide. The combination of said Bacillus strain and fructooligosaccharide when applied to soil that already contains insoluble (or hardly soluble) phosphates, leads to lowering the pH of soil and increasing the phosphate availability in soil. Said Bacillus strain produces organic acids in the soil which results in an increase in solubilization of the phosphorus for plant uptake. Fructooligosaccharide has a dual advantage. It aids the growth of said Bacillus strain as well as promotes production of organic acids by the bacteria. Use of fructooligosaccharide was found to significantly increase the specific microbial count such that at the time of inoculation the amount of said Bacillus strain reaches the threshold number of bacteria that is required to obtain a plant response. Also, use of fructooligosaccharide maintains the necessary number of viable microbial cells in good physiological condition for an acceptable period. Thus, the disclosed composition has a synergistic effect.
The strain of Bacillus siamensis having the deposit accession number MCC 0201 was isolated from soil samples taken in the vicinity of Chandoli Budruk, off Pune-Nashik Highway, Maharashtra. The Applicant has obtained approval for this access from National Biological Authority under Ref # 1177. A pure culture of above mentioned strain of Bacillus siamensis was deposited on February 6, 2020 under terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure at Microbial Culture Collection (MCC) in National Centre for Cell Science (NCCS).
Production of Bacillus strain may be achieved by growing inoculum in nutrient broth containing peptone-10g/l, beef extract-10g/l, sodium chloride- 5 g/l, pH -7.3 and growing at 37OC at 120 RPM for 24 hours.
In accordance with an embodiment, the composition comprises 10^6 to 10^10 cfu of said Bacillus strain per unit of the composition. Preferably, the composition comprises said Bacillus strain in an amount of 10^8 to 10^9 cfu per unit of the composition. For composition in solid form, the composition preferably comprises of 10^6 to 10^9 cfu of said Bacillus strain per gram of the composition. For composition in liquid form, the composition preferably comprises of 10^7 to10^10 cfu of said Bacillus strain per milliliter of the composition.
In accordance with an embodiment, the composition comprises of fructooligosaccharide in an amount of 55-95 % w/w of the composition.
In accordance with an embodiment, said Bacillus strain is coated with fructooligosaccharide. Coating said Bacillus strain with fructooligosaccharide increases the availability thereof to the bacteria. In accordance with an embodiment, said composition comprises Bacillus strain and fructooligosaccharide in a weight ratio ranging between 1:1 to 1:5.
In accordance with an embodiment, the composition comprises a phosphorus source in an amount of 0.5 to 1 % w/w of the composition. Any source of phosphorus that is capable of being solubilized by the deposited strain may be used. In accordance with an embodiment, the one or more phosphorus sources are selected from a group consisting of rock phosphate, monoammonium phosphate, diammonium phosphate, monocalcium phosphate, super phosphate, triple super phosphate, and/or ammonium polyphosphate. In accordance with yet another embodiment, the phosphorus source is an organic fertilizer.
In accordance with an embodiment, the composition comprises of micronutrients such as Zinc, Boron, Copper, Iron, Manganese, or Molybdenum. The micronutrients may be present in the composition alone or in combination in an amount of 10 to 90% w/w of the composition.
In accordance with an embodiment, the composition comprises one or more of known pesticides, acaricides, insecticides and/or nematicides.
In accordance with an embodiment, the composition disclosed herein may comprise one or more optional ingredients. Non- limiting examples of optional ingredients include one or more biologically active ingredients, beneficial microrganisms, biostimulants, preservatives, polymers, wetting agents, surfactants, bulking/carrier agents like peat, perlite, charcoal, kaolin, sand, husks, agar, trehalose, glycerol, sugars, flours etc. or combinations thereof.
In accordance with an embodiment, said composition is in the form of a liquid, a slurry, a solid, or a powder (wettable powder or dry powder). In another embodiment, the composition is in the form of a seed coating.
A method for increasing phosphate availability in soil is also disclosed. In accordance with an embodiment, said method comprises introducing in soil said composition in powder form an amount of 1-30kg/acre. Preferably, said composition is applied to soil in an amount of 15 kg/ acre. In accordance with an alternate embodiment, said method comprises introducing in soil said composition in liquid form in an amount of 0.1-3 liters /acre. Preferably, said composition is applied to soil in an amount of 1.5 liters per acre.
A method for preparing the disclosed composition is also disclosed. Said method comprises a simple mixing of fructooligosaccharide and the Bacillus strain. When the composition comprises other optional ingredients, the composition is prepared by mixing all the ingredients except the Bacillus strain, sterilizing the blend by irradiation, autoclaving, heat treatment (as applicable), or any other appropriate treatment to remove contaminating microorganisms followed by mixing with the Bacillus strain by simple mixing under sterile or controlled environment. In accordance with an embodiment, optional ingredients include one or more biologically active ingredients, beneficial microorganism’s, biostimulants, preservatives, poly. ers, wetting agents, surfactants, bulking/carrier agents like peat, perlite, charcoal, kaolin, sand, husks, agar, trehalose, glycerol, sugars, flours etc. or combinations thereof.

Specific Embodiments Are Described Below
A composition for increasing phosphate availability in soil, the composition comprising an isolated strain of Bacillus siamensis having the deposit accession number MCC 0201; and fructooligosaccharide.
Such composition(s), wherein said Bacillus strain is coated with fructooligosaccharide.
Such composition(s), wherein the composition comprises 10^6-10^10 cfu per unit of said Bacillus strain.
Such composition(s), wherein the composition comprises fructooligosaccharide in an amount of 55 to 95 % w/w of the composition.
Such composition(s), further comprising a phosphorus source in an amount of 0.5 to1 % w/w of the composition.
Such composition(s), further comprising a micronutrient in a concentration of 10-90 % w/w of the composition, said micronutrient being selected from a group consisting of Zinc, Boron, Copper, Iron, Manganese, or Molybdenum.
Such composition, further comprising one or more of pesticides, acaricides, insecticides and/or nematicides.
In order that this invention may be better understood, the following examples are set forth. These examples are for the purpose of illustration only and the exact compositions, methods of preparation and embodiments shown are not limiting of the invention, and any obvious modifications will be apparent to one skilled in the art.
Examples

Example 1:

A. Isolation of Bacillus strain having the deposit accession number - MCC 0201

The strain of Bacillus siamensis having the deposit accession number- MCC 0201 was isolated from soil samples taken in the vicinity of Chandoli Budruk, off Pune-Nashik Highway, Maharashtra. The isolation was carried out using serial dilution method on nutrient agar, Pikovaskys agar (PA). Pure bacterial colonies isolated from press mud was further screened for their phosphate solubilizing activity in vitro using Pikovskaya agar. Quantification of P-solublisation was carried out by measuring the zone of clearance for the phosphate solubilizing bacteria isolates after spotting them in the center of PA plates and incubating at 370C for 2-3 days. Microorganisms if they are capable to solubilize the phosphate from precipitated calcium phosphate in medium they produce clear zone around the colonies. The isolates found positive for P-solubilization were selected and purified by sub-culturing each one 5-6 times on nutrient agar plates. Glycerol stocks for the isolates were prepared for storage at -700C. Based on the zone of clearance, an isolate POT1 was finalized and selected for further analysis.
B. Identification of isolate- POT1
Chromosomal DNA was extracted by using spin column kit (Hi Media, India). Bacterial 16S rRNA gene (1500 bp) was amplified using polymerase chain reaction in a thermal cycler followed by purification using Exonuclease I Shrimp Alkaline Phosphatase. Purified amplicons were sequenced by Sanger method in ABI 3500xL genetic analyser ( Life technologies USA).Sequencing files (.ab1) edited using CHROMASLITE(Version 1.5) and further analysed by Basic Local Alignment Search Tool(BLAST) with closest culture sequence retrieved from the National Centre for Biotechnology Information (NCBI) database were used to find regions of local similarity between sequences compares nucleotide or protein sequences to sequence databases and calculate the statistical significance of matches (Gertz, 2005). Please see Table 1.

Target Sequence Type of Analysis Primer name Results

Species
(Gene Accession) Identity (%)
Seq288_POT1_NC061219B Identification of 16S rRNA gene

++

Applicant’s strain

907R_530F (1244bp) KY643639

Bacillus siamensis KCTC 13613 99 (1237/1241 bp)
KY694464

Bacillus velezensis 99
(1239/1245 bp)

Table 1

C. Growth curve analysis of POT-1

The POT1 was grown in a specially designed medium with 0.2 % fructooligosaccharide (FOS) as sole carbon source and reduced nitrogen content (to restrict excessive growth). The growth was measured by optical density (OD) at 600 nm in 18 hours’ incubation period. Additionally, five independent experiments were carried out in triplicates using Glucose, Inulin, Fructose, Free Sugar-FOS (95% of oligofructose and 5% of other sugars-sucrose 4%,Fructose 0.5%,Glucose 0.2%, Glycerol 0.2%, Arabitol 0.2%) and Tryptone. The growth results have been tabulated in Table 2 below.

% Concentration OD (600nm)
(Expt.1)* OD (600nm)
(Expt.2)** OD (600nm)
(Expt.3)***
0.25%FOS 0.495 1.4755 1.293
0.25%FOS 0.57 1.7865 1.3455
0.25%FOS 0.472 0.96 1.1725

0.25%Glucose 0.013 0.333 0.8015
0.25%Glucose 0.003 0.466 0.745
0.25%Glucose 0.017 0.435 0.481

0.25%Inulin 0.027 0.09 0.0315
0.25%Inulin 0.022 0.003 0.015
0.25%Inulin 0.022 0.092 0.028

0.25% Fructose ND 0.298 0.512
0.25% Fructose ND 0.345 0.415
0.25% Fructose ND 0.472 0.488

Free sugar-FOS 0.0135 0.255 0.552
Free sugar-FOS 0.019 0.283 0.611
Free sugar-FOS _ _ 0.895

0.1% Tryptone 0.022 0.001 0.018
0.1% Tryptone 0.022 0.065 0.018
0.1% Tryptone 0.022 0.019 0.01
Table 2
* Inoculum OD: 1.512
** Inoculum OD: 2.523
***Inoculum OD: 2.536
Observation: It was observed that the medium containing fructooligosaccharide as substrate showed higher cell density compared to glucose, Inulin, Fructose and Tryptone
D. Analysis of total carbohydrate utilization:
For determination of extent of fructooligosaccharide and glucose utilized by POT-1, High performance liquid chromatography (HPLC)were carried out as per the standard optimized method for fructooligosaccharide analysis. The results (in Table 3) showed that fructooligosaccharide is being 100% utilized whereas the utilization of glucose is around 60%.

Values Glucose Fructooligosaccharide
Sample 1 Sample 2 Average Sample 1 Sample 2 Average
% substrate added in the media 0.25 0.25 0.25 0.25 0.25 0.25
Estimated 0.22 0.26 0.25 0.23 0.24 0.235
% remained at 18 hours 0.14 0.16 0.15 0 0 0
% utilized 65 62 63 100 100 100
Table 3
Observation: It was found that fructooligosaccharide was efficiently utilized by the Bacillus strain when compared with glucose after 18 hours.

E. Analysis of total organic acid production by disclosed Bacillus strain

Ingredients Gms / Litre
Yeast extract 0.5
Dextrose 10
Calcium phosphate 5
Ammonium sulphate 0.5
Potassium chloride 0.2
Magnesium sulphate 0.1
Manganese sulphate 0.0001
Ferrous Sulphate 0.0001

Table 4
For determining the extent of organic acid production, the POT1 was grown for 10 days at 30° C in the Pikovaskys growth medium with the composition as given in Table 4. For this study, the fructooligosaccharide content and glucose content in the medium were maintained at 1% (10 g/L). The POT1 was inoculated and organic acids produced were measured in the spent medium after 10 days of growth using HPLC. The results have been summarized in Table 5 below.
HPLC UV Data with Aminex column
Organic Acid Media with glucose (mg/L) Media with FOS (mg/L)
Sample1 Sample 2 Sample
3 Average Average Sample1 Sample2 Sample3
Identified based on Reference standard confirmation
Citric - - - - 198 205 193 197
Succinic 21 20 40 27 140 139 151 130
Lactic 90 89 86 88 77 76 79 77
Acetic 87 111 114 104 1379 1328 1475 1335
Propionic 96 35 34 55 2751 2143 3858 2253
Identified based on the separation rationale of the column
Acrylic 103 104 105 104 1809 1880 1795 1752
Isobutyc 834 (IV) 925 (IV) 833 (IV) 864 1335 1411 1238 1356
Hexanoc 1053 1057 998 1036 465 455 473 467
Table 5
Observation: It was observed that with 1% fructooligosaccharide as substrate, 0.74% of organic acids were produced with Propionic, Acetic, Acrylic and Isobutryric acids being the major constituents. While, with 1% glucose as substrate, only 0.22% of organic acids were produced with Hexanoic, Isobutryric and acetic acids being the major constituents.

F. pH study
The above samples incubated in Pikovaskys broth were tested for resultant pH. Table 6 below shows the observation of pH study.

Source pH
FOS-1 5.05+.02
FOS-2 5.08+.02
FOS-3 5.02+.03

Glucose-1 5.81+0.1
Glucose-2 5.49+0.1
Glucose-3 5.81+0.2

Control-F0S 6.06+0.1
Control-F0S 6.05+0.1

Control-Glucose 5.97+0.1
Control-Glucose 5.96+0.2
Table 6
Observation: It was observed that with fructooligosaccharide as substrate, a lower pH was obtained as compared to that with glucose.

Industrial Applicability

The disclosed composition finds application as an additive for soil for increasing the phosphate availability for plant uptake. The present inventors have found that the disclosed composition when introduced in soil is able to increase the availability of phosphate for plants by producing higher amount of low molecular weight organic acids such as propionic, acetic, lactic, citric, succinic, acrylic and hexonic which solubilizes the insoluble phosphorous.

Higher production of low molecular weight organic acids has a positive impact on the soil by lowering the pH of soil and increasing chelation, exchange reactions, and polymeric substances formation. Herein, the hydroxyl and carboxyl groups of the acids chelate the cations bound to phosphate, thereby converting it into soluble forms. This leads to the increased solubility and availability of phosphorus.

The disclosed composition further transforms organic phosphate into utilizable form through a process of mineralization. Further, the disclosed composition results in increased microbial flora and microbial activity. Additionally, production of higher amount of organic acids inhibit/ control plant pathogens.

The disclosed composition can be used in inorganic/ organic fertilizers, seed coating.