Phosphorus-dissolving fungus, fungicide and application thereof

Abstract

The application discloses a phosphorus-dissolving fungus, a bacterial agent and application thereof, and belongs to the field of environment and agricultural biotechnology. The phosphorus-dissolving fungus is classified and named as Paecilomyces variotii Penicillium steckii ), which is preserved in the China General Microbiological Culture Collection Center on January 8, 2025, and the preservation number is CGMCC NO:41749. The strain has a phosphorus-dissolving amount of more than 700 mg / L, a total concentration of secreted organic acid of more than 5000 mg / L, and a concentration of secreted exopolysaccharide of 5.68 mg / L. The strain directly dissolves insoluble phosphorus by continuously secreting organic acid at normal temperature and pressure, and the secreted exopolysaccharide delays the reprecipitation of soluble phosphorus into calcium phosphate, thereby realizing a double mechanism breakthrough of “in-situ dissolution-in-situ fixation prevention”. The application fundamentally avoids the problems of strong acid pollution in chemical methods and high energy consumption in physical methods, and significantly improves the utilization efficiency of phosphorus and the soil health status.

Description

Technical Field

[0001] This invention belongs to the field of environmental and agricultural biotechnology, specifically relating to a phosphate-solubilizing fungus, a fungal agent, and its application. Background Technology

[0002] Phosphorus is an essential macronutrient for plant growth and development. In industrial and agricultural production, to supplement the insufficient available phosphorus in the soil, the technology of processing and converting natural insoluble phosphates (such as phosphate rock powder) is widely relied upon. Currently, the mainstream technologies all fall under the categories of physical or chemical processes, and there are significant technological bottlenecks and environmental costs.

[0003] Chemical acidification is the most widely used industrial method. Its core is to produce water-soluble phosphate fertilizers (such as superphosphate) by reacting strong acids such as sulfuric acid and phosphoric acid with phosphate rock powder. Although this method is effective, it has the following drawbacks: 1) The process relies on large-scale chemical plants and equipment resistant to strong acid corrosion, resulting in high fixed asset investment and raw material costs; 2) The production process generates a large amount of solid waste (such as phosphogypsum, approximately 5 tons of phosphogypsum are generated for every ton of phosphoric acid produced), which occupies land and poses risks of heavy metal leaching and acidic water pollution; 3) Strong acid fertilizers applied to the soil can easily lead to localized acidification, damaging the soil microbial community and structure.

[0004] Physical pulverization, as an alternative approach, aims to reduce the particle size of phosphate rock to the micron or nanometer level using ultrafine pulverization equipment, thereby increasing its specific surface area and enhancing its reactivity in soil. However, this method does not fundamentally alter the chemical inertness of phosphate rock, and the dissolution and release rate remains slow. More importantly, according to the energy consumption principle of pulverization, the energy required to reduce the particle size of a material by one order of magnitude increases exponentially (up to approximately 100 times), resulting in extremely low energy efficiency and poor economic feasibility for this method.

[0005] Furthermore, both chemically produced soluble phosphate fertilizers and physically refined phosphate rock powder face the fundamental problem of phosphorus re-fixation after being applied to the soil. Water-soluble phosphate ions rapidly combine with metal cations such as calcium, iron, and aluminum in the soil, re-forming insoluble phosphates. This results in the utilization rate of phosphate fertilizer typically being only 10% to 25% in the current season, an inherent defect that existing technologies cannot overcome.

[0006] In summary, existing mainstream technologies suffer from common problems such as high pollution, high energy consumption, low utilization rate, and incompatibility with soil ecology. Therefore, developing an environmentally friendly alternative technology that can efficiently and continuously activate insoluble phosphorus under normal temperature and pressure, effectively prevent phosphorus redeposition in soil, and is therefore urgently needed in agriculture and environmental protection. Utilizing the bio-phosphorus solubilization of microorganisms (phosphate-solubilizing bacteria) presents a highly promising new approach to solving these problems. Summary of the Invention

[0007] In view of this, the purpose of this invention is to provide a phosphorus-solubilizing fungus, a fungal agent, and its application. The QF1 fungus and its fungal agent provided by this invention improve the utilization efficiency of phosphorus by continuously secreting organic acids at room temperature and pressure to directly dissolve insoluble phosphorus, thus providing a novel biological solution for the sustainable utilization of phosphorus resources.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a phosphate-solubilizing fungus, classified under QF1 as *Penicillium fimbriatum* (…). Penicillium steckii It was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 41749.

[0009] Furthermore, the phosphorus solubility reached over 700 mg / L; the total concentration of secreted organic acids exceeded 5000 mg / L; and the concentration of secreted extracellular polysaccharides reached 5.68 mg / L.

[0010] Secondly, the present invention provides a phosphate-solubilizing agent, comprising the aforementioned phosphate-solubilizing fungi.

[0011] Thirdly, the present invention provides the application of the above-mentioned phosphate-solubilizing fungi or phosphate-solubilizing agents in long-acting phosphate solubilization.

[0012] Furthermore, the phosphate-solubilizing fungi continuously secrete organic acids under normal temperature and pressure conditions, directly driving the acidolysis and complexation of insoluble calcium phosphate.

[0013] Furthermore, the phosphate-solubilizing fungi secrete extracellular polysaccharides in the later stages of phosphate solubilization, which can delay the reprecipitation of soluble phosphorus into calcium phosphate.

[0014] It contains at least the following beneficial technical effects: The QF1 and its bacterial agent provided by this invention directly dissolve insoluble phosphorus by continuously secreting organic acids at room temperature and pressure, and delays the reprecipitation of soluble phosphorus into calcium phosphate by secreting extracellular polysaccharides in the later stage of action. This achieves a breakthrough in the dual mechanism of "in-situ dissolution-in-situ anti-fixation", which not only fundamentally avoids the strong acid pollution of chemical methods and the high energy consumption of physical methods, but also significantly improves the utilization efficiency of phosphorus, providing a brand-new biological solution for the sustainable utilization of phosphorus resources. Attached Figure Description

[0015] Figure 1 The images shown are morphological observations and scanning electron microscope (SEM) images of the target strain provided in Example 1 of this invention; where A: colony morphology image; B: lactic acid cotton blue staining image; C: scanning electron microscope image.

[0016] Figure 2 The results of the determination of phosphorus solubility and organic acid content of QF1 in Example 2 of the present invention are shown.

[0017] Figure 3 This is the result of the determination of QF1 extracellular polysaccharides in Example 2 of the present invention. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25 ± 2 ℃.

[0024] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0025] Example 1 Isolation and identification of strains S1. Sample collection: Soil samples were collected from Xilingol League, Inner Mongolia (43°57'48"N, 116°4'9"E); S2. Enrichment culture: Following the soil fungal isolation procedure, the soil sample from step S1 was cultured at 30 °C and 180 r / min for 30 min. S3. Isolation and Culture: Under aseptic conditions, the soil suspension cultured in step S2 is serially diluted to obtain 10... -2 10 -3 10 -4 10 -5 and 10 -6 Soil suspensions with dilution gradients were prepared. 100 μL of bacterial suspensions of different concentrations were spread onto PVK solid plates containing chloramphenicol and streptomycin, with three replicates for each gradient. The plates were incubated upside down at 30 °C for 3–9 days. Single colonies with good growth, typical characteristics, and large, prominent phosphate-solubilizing zones were picked using an inoculation loop and further purified by streaking on PVK solid medium to obtain pure cultures of the target strain, which were then preserved.

[0026] The isolation medium was PVK liquid medium (1 L formulation): glucose 10.0 g, NaCl 0.3 g, MgSO4·7H2O 0.3 g, KCl 0.3 g, (NH4)2SO4 0.5 g, Ca3(PO4)2 5.0 g, yeast extract 0.5 g, MnSO4·H2O 0.03 g, FeSO4·7H2O 0.03 g, streptomycin 50 mg, chloramphenicol 50 mg, pH 7.1~7.2, and 18 g of agar was added to the solid medium.

[0027] The preservation medium was LB medium (1 L formula): 10 g peptone, 5 g yeast extract, 10 g NaCl, pH 7.0~7.2, with 18 g agar added to the solid medium.

[0028] A phosphate-solubilizing fungal strain, designated QF1, was successfully isolated from environmental samples using the method described above. This strain exhibits strong phosphate-solubilizing ability.

[0029] S4. Strain identification: Strain QF1 (e.g., obtained through steps S2 and S3) is then identified. Figure 1 As shown in the figure, it was identified as the target strain; the phosphorus solubility of the strain was determined by the molybdenum antimony colorimetric method. After 6 days of culture, the phosphorus solubility of strain QF1 was 739.6 mg / L, indicating that strain QF1 has a strong phosphorus solubility.

[0030] The following is the identification of the QF1 strain: Colony characteristics of strain A.QF1 A phosphate-solubilizing fungus, specifically strain QF1, has colony characteristics of being round with a raised center, a relatively smooth surface, and an overall grayish-green color with a lighter central area. The colony edges are clearly defined with a slight ring-like structure, and the overall morphology is relatively uniform. Figure 1 ).

[0031] ITS region sequence analysis of B.QF1 strain The ITS region of QF1 was sequenced, and the resulting sequence was compared with the NCBI nucleotide database using BLAST. The highest similarity was 100%, and it was identified as Penicillium fimbriatum. Penicillium steckii Currently, there are no literature reports on the phosphate-solubilizing ability of *Penicillium fimbriae*. Therefore, the screening of strain QF1 enriches the genetic resources of wild-type phosphate-solubilizing bacteria and expands the reserve pool for whole-genome breeding of phosphate-solubilizing bacteria. The gene sequence of QF1 has been submitted to the GenBank database, accession number PQ835802.

[0032] Example 2 Phosphorus solubilization mechanism research To verify and utilize the phosphate-solubilizing mechanism of the QF1 strain described in Example 1, liquid culture and quantitative detection of metabolites are required. The specific steps are as follows: Using a sterile inoculation loop, scrape the activated spores and inoculate them into an Erlenmeyer flask containing 100 mL of NBRIP liquid medium (formula: glucose 10.0 g, Ca3(PO4)2 5.0 g, MgCl2·6H2O 5.0 g, MgSO4·7H2O 0.25 g, KCl 0.2 g, (NH4)2SO4 0.1 g, diluted to 1 L with distilled water, pH 7.0). The spore inoculation amount is approximately 1 × 10⁻⁶. 6 cells / mL ~ 1×10 8 Cells / mL, cultured at 30 ℃ and 160 r / min for 14 days with shaking.

[0033] Determination of phosphorus solubility During the incubation period, aseptically take 5 mL of culture medium every 2 days, centrifuge at 4 ℃ and 10000 r / min for 10 min, and obtain the supernatant. Accurately pipette 200 μL of the supernatant into a 50 mL volumetric flask and dilute with approximately 15 mL of distilled water. Add 1 drop of 2,6-dinitrophenol indicator, and add dilute sulfuric acid until the solution just turns slightly yellow. Then add 5 mL of molybdenum antimony colorimetric reagent (prepared fresh each time: dissolve 1.5 g of ascorbic acid in 100 mL of molybdenum antimony reagent stock solution), and bring the volume to 50 mL with distilled water, then shake well. After standing at room temperature for 30 min, measure the absorbance value at a wavelength of 720 nm using a spectrophotometer.

[0034] Organic acid determination The supernatant obtained from centrifugation in the "Phosphorus Dissolution Determination" was filtered through a 0.22 μm aqueous filter membrane. Analysis was performed using high-performance liquid chromatography (HPLC). Specific conditions were: column: C18 reversed-phase column; mobile phase: 0.02 mol / L potassium dihydrogen phosphate buffer solution at pH 2.6 mixed with methanol at a volume ratio of 99:1; flow rate: 0.5 mL / min; column temperature: 30 ℃; detection wavelength: 210 nm; injection volume: 10 μL. The retention times of each peak in the sample chromatogram were compared with the retention times of organic acid standards such as oxalic acid, citric acid, and malic acid for qualitative analysis. The concentration of each organic acid was quantitatively calculated by calculating the peak area and substituting it into the corresponding standard curve.

[0035] Extracellular polysaccharide assay Take 5 mL of culture medium, centrifuge at 10000 r / min for 15 min at 4 °C, collect the supernatant, heat in a boiling water bath for 10 min to denature the protein, cool, and centrifuge at 10000 r / min for 15 min at 4 °C to remove the precipitate. Add 3 volumes of pre-cooled anhydrous ethanol to the supernatant and let it stand in a refrigerator at 4 °C for 24 h to precipitate the polysaccharide. Centrifuge at 10000 r / min for 15 min at 4 °C to collect the precipitate, dry at 50 °C to obtain crude extracellular polysaccharide. Dissolve the crude polysaccharide in 1 mL of distilled water. Take 0.2 mL of this solution, add distilled water to 2.5 mL, add 6.0 mL of anthrone sulfate reagent (2% anthrone in concentrated sulfuric acid solution), heat in a boiling water bath for 10 min, and cool to room temperature. Measure the absorbance at 620 nm using a spectrophotometer. Substitute the absorbance value into a pre-plotted glucose standard curve to calculate the content of extracellular polysaccharide (expressed as glucose equivalent).

[0036] Effect description See Figure 2 From day 3 onwards, *Penicillium fimbriatum* began to secrete organic acids significantly, and the amount of phosphorus dissolved also increased rapidly, indicating that acidification was the main driving force for phosphorus dissolution. On days 6-7, both the concentration of organic acids and the amount of phosphorus dissolved reached their peak (approximately 700-750 mg / L for phosphorus dissolution and over 5000 mg / L for organic acids), indicating that organic acid secretion is one of the key metabolic processes for calcium phosphate dissolution in this strain. Organic acids lower the pH of the culture system and complex Ca²⁺. + In addition, it accelerates the conversion of insoluble calcium phosphate to soluble phosphorus through methods such as promoting mineral acid hydrolysis. In the later stage of cultivation (after 7 days), as the level of organic acid secretion decreased, the amount of phosphorus dissolved also decreased significantly, indicating that a continuous supply of organic acids plays an important role in maintaining a high level of phosphorus solubility, and a single phosphorus solubility mechanism is difficult to support the continuous release of phosphorus in the later stage.

[0037] See Figure 3The extracellular polysaccharides exhibited a dynamic pattern of "fluctuation-peak-decline" throughout the culture process, reaching a peak value (5.68 mg / L) on day 10. Combined with... Figure 2 It is evident that extracellular polysaccharides play a regulatory and stabilizing role in the later stages of phosphorus solubility, rather than directly driving mineral dissolution. Extracellular polysaccharides can complex with Ca²⁺. + The formation of a colloidal structure affects Ca²⁺. + With PO4³ - The recombination process of phosphorus in *Penicillium fimbriatum* can, to some extent, delay the redeposition of dissolved phosphorus. The phosphorus-dissolving process in *Penicillium fimbriatum* is not the result of a single mechanism, but rather a combination of an early mineral dissolution process dominated by organic acids and a later regulation process involving extracellular polysaccharides. This phased synergistic mechanism helps improve the dissolution efficiency of calcium phosphate and delays the precipitation of phosphorus in the culture system.

[0038] As can be seen from the above experiments, the Penicillium fimbriatum used in this invention can simultaneously secrete organic acids and extracellular polysaccharides during the dissolution of insoluble calcium phosphate. The synergistic effect of the two significantly improves the phosphorus dissolution efficiency and stability.

[0039] Experimental results showed that on days 6-7 of cultivation, the dissolved phosphorus concentration reached over 700 mg / L, far exceeding the level of natural dissolution; at this time, the total organic acid concentration exceeded 5000 mg / L, proving that the strain directly drives mineral dissolution through the secretion of organic acids. In the later stage of phosphorus dissolution (days 9-10), the extracellular polysaccharide concentration reached 5.68 mg / L, through complexation with Ca²⁺. + It also forms a colloidal barrier, delaying the redeposition of soluble phosphorus.

[0040] Compared with existing chemical acidification and physical pulverization methods, the fungal phosphate-solubilizing mechanism of this invention can efficiently dissolve insoluble phosphates under normal temperature and pressure, without the addition of strong acids and with low energy consumption. It can also delay phosphorus refixation through extracellular polysaccharides, which significantly improves the effectiveness and sustainability of phosphorus, achieving a dual improvement effect of "dissolution + anti-fixation".

[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A phosphate-solubilizing fungus, characterized in that, The phosphate-solubilizing fungus, classified as Penicillium steckii in the QF1 classification, was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 41749. The phosphorus solubility reaches over 700 mg / L; the total concentration of secreted organic acids exceeds 5000 mg / L; and the concentration of secreted extracellular polysaccharides reaches 5.68 mg / L.

2. A phosphate-solubilizing agent, characterized in that, Includes the phosphate-solubilizing fungi as described in claim 1.

3. The application of the phosphate-solubilizing fungus of claim 1 or the phosphate-solubilizing agent of claim 2 in long-acting phosphate solubilization.

4. The application according to claim 3, characterized in that, The phosphate-solubilizing fungi continuously secrete organic acids under normal temperature and pressure conditions, directly driving the acidolysis and complexation of insoluble calcium phosphate.

5. The application according to claim 3, characterized in that, The phosphate-solubilizing fungi secrete extracellular polysaccharides in the mid-to-late stages of phosphate solubilization, which delays the reprecipitation of soluble phosphorus into calcium phosphate.

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