Novel paenibacillus with biomacromolecule degradation capacity and nitrogen fixing and phosphorus dissolving effects and application of novel paenibacillus

Abstract

The invention relates to novel paenibacillus with biomacromolecule degradation capacity and nitrogen fixation and phosphorus solubilization effects and application of the novel paenibacillus, the strain is identified as a novel strain of paenibacillus through multi-phase classification, and the novel paenibacillus has the capacity of degrading biomacromolecules such as starch, cellulose, casein, chitin and lecithin. The method can be applied to degradation of related carbohydrates, proteins and lipid substances and preparation of biological enzymes; the bacillus subtilis has the advantages that the bacillus subtilis can be used for preparing the bacillus subtilis, has the phosphorus dissolving capacity of dissolving organic phosphorus and inorganic phosphorus, has nitrogenase and beta-1, 3-glucanase and the activity of generating IAA (Indoleacetic Acid), can play a probiotic role on crops by fixing nitrogen, dissolving phosphorus and inhibiting pathogenic bacteria, can grow in an environment with the salinity not higher than 4.5%, and has certain salt-tolerant activity. Due to multiple functions, the strain can be used for preparing and applying biological nitrogen fertilizers, phosphate fertilizers and crop probiotics, so that the strain plays an important role in crop growth and green agricultural production.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a novel Bacillus-like bacterium with the ability to degrade biological macromolecules and to fix nitrogen and solubilize phosphorus, and its applications. Background Technology

[0002] With the increasing demand for sustainable agricultural development globally, soil degradation, environmental pollution, and agricultural product safety issues caused by the long-term use of chemical fertilizers and pesticides are becoming increasingly prominent. Soil microecological imbalance and nutrient deficiency have become significant bottlenecks to the sustainable development of modern agriculture, even affecting the nutritional levels of agricultural products and ultimately impacting human health. According to statistics from the Food and Agriculture Organization of the United Nations, approximately 30% of the world's arable land suffers from varying degrees of soil degradation, necessitating the development of environmentally friendly and sustainable agricultural production technology systems.

[0003] Soil micro-ecosystems, as the core carriers of agricultural production, directly determine soil fertility, crop resistance, and agricultural product quality. Microorganisms are a crucial component of these micro-ecosystems and a vital driving force for the natural cycle of matter. The restoration or biofortification of rhizosphere microbial function is receiving increasing attention in soil nutrition and health, and in green agricultural production. The research and development of various microbial agents and fertilizers play a significant role in driving the cycling of key plant nutrients such as nitrogen, phosphorus, and potassium, increasing soil organic matter content and availability after macromolecular degradation, and enhancing pathogen resistance, continuously promoting high-quality green agricultural development. The core competitiveness of high-quality microbial agents or fertilizers depends on the performance of functional strains, i.e., on high-performance microbial strains. Therefore, the development and application of agricultural probiotic microbial strains has always been a research hotspot and core focus in the field of modern agricultural biotechnology.

[0004] Among numerous agricultural probiotic microbial resources, the genus *Paenibacillus* has attracted widespread attention from the scientific and industrial communities due to its functional diversity and excellent environmental adaptability. First reported in 1973, this genus currently contains over 300 species. Its cells are Gram-positive, and it lives in facultative anaerobic or strictly aerobic environments, adapting to various ecological environments such as soil, plant rhizosphere, and water bodies. Different *Paenibacillus* species have been reported to possess different functions, such as hydrolysis of starch and casein, and some antibacterial abilities, thus showing great promise for applications in plant disease resistance and crop probiotics. However, existing *Paenibacillus* resources still have certain functional limitations, such as insufficient functional diversity and low viability. Therefore, screening and discovering new *Paenibacillus* species with complex probiotic functions is of significant theoretical and practical value for promoting the upgrading and iteration of agricultural microbial agents and supporting the high-quality development of green agriculture, and is also the core starting point of this invention. Summary of the Invention

[0005] The purpose of this invention is to provide a novel Bacillus-like organism with the ability to degrade biological macromolecules and to fix nitrogen and solubilize phosphorus, and its applications.

[0006] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a novel Bacillus sp. TH7-28, which has the ability to degrade biological macromolecules and fix nitrogen and solubilize phosphorus. It was isolated and screened from freshwater lake sediments and deposited at the China General Microbiological Culture Collection Center on December 4, 2025, with the culture collection number CGMCC No. 36894.

[0007] The 16S rRNA gene sequence of this strain has been submitted to the GenBank database, accession number PQ816947; the main biological characteristics of this bacterium are Gram-positive, rod-shaped cells, positive catalase reaction, positive oxidase reaction, and salt tolerance of 4.5% (w / v, NaCl).

[0008] Based on the results of multiphasic taxonomic identification, including comprehensive morphological, physiological and biochemical characteristics, genotypic and phylogenetic analysis, *Bacillus th7-28* is a new species of the genus *Bacillus*.

[0009] Its genome contains genes encoding amylase, cellulase, protease, phospholipase, chitinase, and β-1,3-glucan, as well as functional genes related to inorganic phosphorus dissolution and transport, and nitrogenase genes.

[0010] Secondly, the present invention provides a method for culturing the above-mentioned Bacillus TH7-28: Bacillus TH7-28 is inoculated into TSA medium and activated at 37 °C; the fresh bacterial cells obtained are inoculated into TSB liquid medium and cultured at 37 °C to form a seed culture; the seed culture is inoculated into TSB liquid medium at a volume ratio of 5% and cultured at 37 °C to obtain a live bacterial culture of TH7-28.

[0011] The TSA medium consists of: 15 g / L tryptone, 5 g / L soybean peptone, 5 g / L sodium chloride, and 15 g / L agar, with a pH of 7.5. For liquid culture, TSB medium is used, which has the same nutrient composition as TSA but does not contain agar.

[0012] Thirdly, the present invention provides a microbial agent containing the aforementioned Paenibacillus sp. TH7-28.

[0013] Fourthly, the present invention provides a microbial agent containing the fermentation product of the aforementioned Paenibacillus sp. TH7-28.

[0014] Preferably, the above-mentioned microbial agent is a solid microbial agent or a liquid microbial agent.

[0015] Fifthly, the present invention provides the application of the above-mentioned Paenibacillus sp. TH7-28 and its agent in the degradation of starch, cellulose, casein, chitin, lecithin and β-glucan.

[0016] In a sixth aspect, the present invention provides the application of the above-mentioned Paenibacillus sp. TH7-28 and its inoculant in promoting plant growth.

[0017] In a seventh aspect, the present invention provides the use of the above-mentioned Paenibacillus sp. TH7-28 and its agent in dissolving inorganic phosphorus and / or degrading organic phosphorus.

[0018] Eighthly, the present invention provides the application of the above-mentioned Paenibacillus sp. TH7-28 and its bacterial agent in the preparation of bio-phosphate fertilizer bacterial agents, bio-nitrogen fertilizer bacterial agents and plant probiotic agents.

[0019] The beneficial effects of this invention are as follows: The strain identified by polyphasic classification is a new species of *Paenibacillus*, possessing the ability to degrade biomacromolecules such as starch, cellulose, casein, chitin, lecithin, and β-glucan. It can be applied to the degradation of related sugars, proteins, and lipids, as well as the preparation of bioenzymes. Simultaneously, it has the ability to dissolve organic phosphorus and degrade inorganic phosphorus, exhibiting nitrogenase activity. It can exert beneficial effects on crops through nitrogen fixation, phosphorus dissolution, inhibition of pathogens, and IAA production. Furthermore, it can grow in environments with salinity not exceeding 4.5%, demonstrating a certain degree of salt tolerance. These multiple functions make this strain suitable for the preparation and application of bio-nitrogen fertilizers, phosphate fertilizers, and crop probiotic agents, thus playing a vital role in crop growth and green agricultural production. Attached Figure Description

[0020] Figure 1 The cell morphology of strain TH7-28 under a scanning electron microscope; Figure 2 Phylogenetic tree of strain TH7-28 based on 16S rRNA gene sequence; Figure 3 This is a circumscribed genome diagram of strain TH7-28; Figure 4 This is a graph showing the biomolecular degradation capacity of strain TH7-28. Figure 5 The graph shows the chitinase and β-1,3-glucanase activities of strain TH7-28. Figure 6 This is a graph showing the phosphate solubility of strain TH7-28. Figure 7 The nitrogenase activity of strain TH7-28 is shown in the graph. Figure 8 This is a diagram showing the IAA production of strain TH7-28. Figure 9 This image shows the germination and growth promotion results of wheat strain TH7-28. Detailed Implementation

[0021] To better illustrate the technical solutions and implementation effects of the present invention, the present invention will be further described below in conjunction with the embodiments, but is not limited to the following embodiments.

[0022] Example 1: Isolation, screening, and physiological and biochemical characteristics of bacterial strains Paenibacillus sp. TH7-28 was isolated from sediments in freshwater bodies. Sediment samples were serially diluted tenfold and plated onto TSA medium. After incubation at 37 °C for 2 days, single colonies were picked and purified using the three-zone streak method to obtain strain TH7-28. The growth conditions of the strain were investigated using TSB as the basal medium, with different temperatures (4, 10, 15, 28, 30, 37, 40, 45, 50, and 60 °C), different pH values ​​(3.0-12.0 in 0.5 pH increments), and different salinities (0-5% w / v, NaCl in 0.5% increments) established. Growth was assessed using OD... 600 Growth concentration was monitored. The activities of catalase and oxidase were determined using hydrogen peroxide and N,N-dimethyl-p-phenylenediamine, respectively. Cells cultured at 37 °C for 24 h using TSB were used to test quinone types, polar esters, and fatty acid types. Quinone types were determined by HPLC, polar esters by two-dimensional thin-layer chromatography on silica gel 60 plates, and fatty acids by the Sherlock Microbial Identification System (MIDI). Other indicators were determined according to the *Handbook of Systematic Identification of Common Bacteria*.

[0023] The results showed that strain TH7-28 had rod-shaped cells with a size of (0.2-0.5) µm × (1.4-2.4) µm (see attached results). Figure 1Gram-positive; colonies are white, round, raised, and 1-2 mm in diameter. Strain TH7-28 can grow at 22-55 ℃, pH 6.0-9.0, and NaCl salinity below 4.5%. It is positive for both catalase and oxidase, has the quinone form MK-7, and its polar lipids mainly contain phosphatidylethanolamine, two amino lipids, and three phospholipids. The main fatty acids (>10%) are anteiso-C15:0 (34.54%), anteiso-C17:0 (19.64%), iso-C16:0 (14.36%), and C16:0 (13.22%).

[0024] Example 2: Identification of the taxonomic position of the fungal species Genomic DNA was extracted from the strain using the SDS-alkali lysis method. The 16S rRNA gene was amplified by PCR using universal primers E. coli 27F and 1492R. After 1% agarose gel electrophoresis, the nucleotide sequence was determined using the Sanger dideoxy chain termination method. The obtained sequences were aligned to the GenBank database, and a phylogenetic tree was constructed using the neighbor-joining method with MEGA 7.0 software. The GenBank accession number for the 16S rRNA gene sequence of strain TH7-28 is PQ816947. The phylogenetic tree based on the 16S rRNA gene is attached. Figure 2 As shown. Strain TH7-28 belongs to the genus Paenibacillus and is related to Paenibacillus oralis KCOM 3021. T and Paenibacillus macerans ATCC 8244 T The two strains are most closely related, with homology of 98.2% and 98.1%, respectively. However, strain TH7-28 is similar to P. oralis KCOM 3021. T And P. maceransATCC 8244 T There are several significant differences among the physiological and biochemical indicators (some results are shown in Appendix Table 1), such as growth conditions, catalase and oxidase activities, carbon source utilization, and GC content. Based on the combined results of phylogenetic analysis, morphological indicators, physiological and biochemical indicators, and genomic differential analysis, this study indicates that TH7-28 is a new species of the genus *Bacillus*.

[0025] Appendix Table 1. Strains TH7-28 and their most relevant species P. oralis KCOM 3021 T And P. macerans ATCC8244 T Differences in physiological and biochemical characteristics feature TH7-28 <![CDATA[ATCC 8244 T ]]> <![CDATA[KCOM 3021 T ]]> Salinity for growth (%) Optimal 0-4.5 (1.5) 0-5.0 (1.0) 0-4.0 (1.0) Optimal pH for growth 6.0-9.0 (7.5) 3.0-9.0 (7.0) 5.5-9.5 (8.0-8.5) Growth temperature (°C; optimum) 20-55 (45) 20-55 (40) 25-45 (35) Indole production + - - Nitrate reduction + - + Oxidase + - - catalase + - + Casein hydrolysis + - - Carbon source utilization glycerin + - + Trehalose - - + Xylose + + - Raffinose + - + GC content (mol%) 52.1 52.5 51.3 Fatty acids (>10%) <![CDATA[anteiso-C 15:0 (34.54%)、antecede-C 17:0 (19.64%)、iso-C 16:0 (14.36%)、C 16:0 (13.22%)]]> <![CDATA[anteiso-C 15:0 (21.28%)、C 16:0 (19.82%)、antecedent-C 17:0 (15.27%)、iso-C 16:0 (12.3%)]]> <![CDATA[anteiso-C 15:0 (43.4%)、C 16:0 (16.6%)、iso-C 16:0 (14.5%)、antecede-C 17:0 (12.4%)]]> Example 3: Genomic genetic characteristics of the strain Strains TH7-28 were cultured using TSB at 37 ℃ and 150 rpm for 12 h. After centrifugation and washing with sterile water, genomic DNA was collected and purified. Insert genome sequencing libraries were constructed from quality-controlled samples using the Illumina NovaSeq 6000 sequencing platform, and paired-end sequencing was performed. Base distribution and quality fluctuations were analyzed for each sequencing cycle, and the base quality, cycle error rate, and base distribution of the samples were analyzed to ensure the quality of the strain's genome library construction and sequencing. The sequenced sequences were then assembled, partially assembled, and optimized. Coding sequence prediction was performed using Glimmer, GeneMarkS, and Prodigal software; tRNA identification was performed using tRNAscan-SE v2.0; rRNA prediction was performed using Barrnap software; and sRNA prediction annotation was performed using the Infernal and Rfam databases. A genome circummap was constructed using Circos software, and the genome length and GC content were calculated. For genomic data that have undergone quality testing and sequence assembly, non-redundant protein databases are used for NR annotation, Swiss-prot and Pfam databases are used for COG, GO, KEGG and other functional annotations, and CAZy database is used for carbohydrate active enzyme annotation.

[0026] The genome circle diagram of strain TH7-28 is attached. Figure 3 The genome of this bacterium is 7,279,269 bp in size, containing 6,281 protein-coding genes, 80 tRNAs, and 5 rRNAs, with a DNA GC content of 52.1%. Strain TH7-28 is most closely related to the genus *P. macerans* ATCC 8244. T And P. oralis KCOM 3021 T The ANI values ​​were 91.00% and 92.85%, respectively, and the DDH values ​​were 44.60% and 52.40%, respectively, which were far lower than the standard of 95% and 70% for the same genus, further confirming that strain TH7-28 is a new species of Bacillus spp.

[0027] Genomic analysis revealed that strain TH7-28 contains a large number of genes related to the metabolism of different types of biological macromolecules. These include genes such as 1,4-β-cellobiosidase cbhA for cellulose degradation, pullulanase nplT for starch degradation, and protease pqqL for protein degradation. Additionally, the genome contains genes for lignin and pectin esterases that degrade lignin and pectin, as well as genes for organophosphate degradation such as N-acyl-phosphatidylethanolamine hydrolase, phospholipase D1 / 2, cardiolipin-specific phospholipase, lysophospholipase, and phospholipase / carboxylesterase.

[0028] Meanwhile, the TH7-28 genome contains genes encoding chitinase and endoglucanase, which inhibit the growth of pathogenic fungi by interfering with cell wall synthesis; genes encoding nitrogenase such as nifH / D / K / E / N; genes related to inorganic phosphorus solubility such as pstS / C / A / B; and genes encoding phosphate starvation-induced proteins such as phoH / R / P / A.

[0029] The aforementioned genomic and genetic background reveals a powerful functional enzyme encoding system. The TH7-28 strain possesses a broad spectrum of biomolecule degradation capabilities, including cellulose, starch, protein, and phosphate esters, as well as plant growth-promoting abilities such as phosphorus solubilization, nitrogen fixation, and inhibition of pathogenic fungal growth.

[0030] Example 4: Degradation activity of strain TH7-28 against starch, protein, cellulose and lecithin Strain TH7-28 was inoculated into TSA medium and cultured at 37°C for 24 hours to activate the cells. Using TSA medium as the basal medium, 1% (w / v) of casein, starch, sodium carboxymethyl cellulose, and lecithin were added respectively. Fresh cells were inoculated into media containing different biomolecules and cultured at 37°C for 2 days. The hydrolysis of casein and lecithin was directly observed by the formation and size of the clear zone. The hydrolysis of starch was observed by spraying the medium containing the cells with iodine solution and then observing the clear zone. The hydrolysis of cellulose was observed by spraying the medium with 1% Congo red solution and then observing the size of the clear zone. Results (see appendix). Figure 4 The results showed that strain TH7-28 had a significant ability to degrade casein, starch, cellulose and lecithin. Example 5: Chitinase and β-1,3-glucanase activities of strain TH7-28 Strain strain TH7-28 was inoculated into TSB medium and cultured at 37°C and 150 r / min for 24 hours. Cells were collected by centrifugation and resuspended in sterile water to allow OD to rise. 600The concentration was 1.0. The resuspended bacterial suspension was inoculated (v / v, 5%) into chitinase fermentation medium (chitosan 10 g, yeast extract 2 g, K2HPO4 0.7 g, KH2PO4 0.3 g, MgSO4 0.5 g, FeSO4·H2O 0.02 g, NaCl 5 g, distilled water to 1 L, pH 7.0, sterilized at 121℃ for 20 min) and β-1,3-glucanase fermentation medium (laminarin 5 g, yeast extract 3 g, K2HPO4 1 g, NaNO3 3 g, KCl 0.5 g, MgSO4 0.5 g, FeSO4·H2O 0.5 g, distilled water to 1 L, pH 7.0, sterilized at 121℃ for 20 min) and cultured at 37℃ and 150 r / min for 5 days. The supernatant was collected, and chitinase and β-1,3-glucanase activities were detected using a chitinase kit and the DNS method, respectively. The results showed that the chitinase activity of strain TH7-28 was 8.12 U / L, and the β-1,3-glucanase activity of strain TH7-28 (on day 3) was 4.46 μg / mL / min (see attached results). Figure 5 This bacterium can effectively degrade chitin and β-1,3-glucan, thereby inhibiting the synthesis of cell walls in related fungi.

[0031] Example 6: Phosphate-solubilizing and nitrogen-fixing activities of strain TH7-28 Strain strain TH7-28 was inoculated into TSA medium and cultured at 37°C for 24 hours to activate the cells. Fresh cells were then inoculated into inorganic phosphorus medium with the following composition: glucose 10 g, MgCl2·6H2O 5 g, MgSO4·7H2O 0.25 g, KCl 0.20 g, (NH4)2SO4 0.10 g, Ca3(PO4)2 5 g, agar 15 g, and deionized water to a final volume of 1 L. The medium was pH 7.0 and sterilized at 115°C for 30 min. The cells were then incubated at 37°C for 3 days, and the size of the clear zone was observed. The results are attached. Figure 6 As shown, strain TH7-28 exhibits a significant ability to dissolve inorganic phosphorus, promoting the dissolution of insoluble inorganic phosphorus. Furthermore, the degradation results on lecithin indicate that strain TH7-28 also possesses the ability to degrade organic phosphorus. Therefore, strain TH7-28 demonstrates a significant effect on enhancing phosphorus bioavailability, contributing to the utilization of phosphorus in soil and other environments.

[0032] The TH7-28 strain was inoculated into TSB medium and cultured at 37°C and 150 r / min for 24 hours. Cells were collected by centrifugation and resuspended in sterile water to allow OD to rise. 600The bacterial suspension was inoculated (v / v, 5%) into Ashby nitrogen-free medium (mannitol 10 g, KH2PO4 0.2 g, MgSO4·7H2O 0.2 g, NaCl 0.2 g, CaSO4·2H2O 0.1 g, CaCO3 5 g, distilled water 1 L, pH adjusted to 7.2-7.4, sterilized at 121℃ for 20 min) and cultured at 37℃ and 150 r / min for 5 days. The supernatant was collected, and nitrogenase activity was detected using a nitrogenase kit. The results showed that the nitrogenase activity of strain TH7-28 was 163.65 U / L (see Appendix). Figure 7 This confirmed the nitrogen-fixing ability of the strain.

[0033] Example 7: IAA production activity of strain TH7-28 The TH7-28 strain was inoculated into TSB medium and cultured at 37℃ and 150 r / min for 24 hours to prepare a seed culture. The seed culture was then inoculated at a ratio of 5% into TSB medium containing 0.2 g / L L-tryptophan and cultured at 37℃ and 150 r / min for 5 days. Samples were taken daily, centrifuged, and the supernatant was used to determine the content of the auxin indoleacetic acid (IAA). 2 mL of the TH7-28 culture supernatant was mixed with an equal volume of Salkowski colorimetric reagent (50 mL 35% HClO4 + 1 mL 0.5 mol / L FeCl3), and incubated at room temperature in the dark for 30 min. A sterile mixture of TSA and Salkowski colorimetric reagent served as a blank control. The concentration of the OD was measured... 530 The absorbance at each concentration was measured using a spectrophotometer, and the IAA yield was calculated based on the standard curve. The results are attached. Figure 8 As shown, strain TH7-28 produced 46.00 mg / L of IAA on day 4.

[0034] Example 8 Plant growth promotion ability of strain TH7-28 Six hundred plump and uniformly sized wheat seeds were selected and soaked in a 1% sodium hypochlorite solution for 10 minutes for surface disinfection. After disinfection, the seeds were thoroughly rinsed several times with distilled water and air-dried on sterile gauze. Strains TH7-28 were prepared into a 1×10⁻⁶ solution with sterile water. 8A bacterial suspension of CFU / mL was used. One hundred wheat seeds were immersed in the bacterial suspension using the soaking method, while the control group was immersed in sterile water. Each treatment was replicated. After soaking at 28 °C for 2 h, the seeds were dried on filter paper for 5 minutes, then transferred to moist gauze, and covered with another layer of moist gauze for germination. The germination rate of the wheat seeds was recorded, and five seedlings were randomly selected from each replicate group for length measurement. The results showed that the germination rate of wheat seeds in the TH7-28 treatment group was 94.33%, which was 5.99% higher than that in the blank control group; the sprout length of wheat in the TH7-28 treatment group was significantly better than that in the blank control group (see Appendix). Figure 9 It has a significant ability to promote wheat growth.

[0035] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A novel Bacillus-like bacterium with the ability to degrade biomolecules and fix nitrogen and solubilize phosphorus, characterized in that, The strain is named Paenibacillus sp. TH7-28 and is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 36894.

2. The method for culturing Bacillus TH7-28 as described in claim 1, characterized in that: Bacillus subtilis TH7-28 strain was inoculated into TSA medium and activated at 37 ℃; the fresh cells obtained from the culture were inoculated into TSB liquid medium and cultured at 37 ℃ to form a seed culture. The seed culture was inoculated into TSB liquid medium at a volume ratio of 5% and cultured at 37 °C to obtain TH7-28 live bacterial culture.

3. A microbial agent, characterized in that, This bacterial agent contains the Bacillus subtilis TH7-28 as described in claim 1.

4. The microbial agent as described in claim 3, characterized in that, This inoculum contains the fermentation product of the aforementioned Bacillus subtilis TH7-28.

5. The microbial agent as described in claim 3 or 4, characterized in that, The microbial agent can be a solid microbial agent or a liquid microbial agent.

6. The use of Bacillus subtilis TH7-28 as described in claim 1 and the bacterial agent as described in any one of claims 3-5 in the degradation of starch, cellulose, casein, chitin, lecithin and β-glucan.

7. The use of the Bacillus subtilis TH7-28 of claim 1 and the bacterial agent of any one of claims 3-5 in promoting plant growth.

8. The use of Bacillus subtilis TH7-28 as described in claim 1 and the bacterial agent as described in any one of claims 3-5 in dissolving inorganic phosphorus and / or degrading organic phosphorus.

9. The use of Bacillus subtilis TH7-28 as described in claim 1 and the bacterial agent as described in any one of claims 3-5 in the preparation of bio-phosphate fertilizer bacterial agents, bio-nitrogen fertilizer bacterial agents, and plant probiotic agents.

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