Acquisition and optimization method of phosphorus-solubilizing fungus

Abstract

The invention discloses a phosphorus-solubilizing fungus obtaining and optimizing method, and belongs to the field of environment and the field of agricultural biotechnology. The method comprises the following steps: collecting a soil sample of Inner Mongolia Tin-Go Union; performing liquid culture on the soil sample, and performing gradient dilution under a sterile condition; the method comprises the following steps: sucking 100 [mu] L of bacterial solutions with different concentrations, coating the bacterial solutions on a PVK solid culture medium added with chloramphenicol and streptomycin, culturing, selecting single colonies with good growth, typical characteristics and large and remarkable phosphate solubilizing circle, continuously carrying out streak purification on the PVK solid culture medium to obtain a target strain pure culture, preserving and identifying to obtain the phosphate solubilizing fungi; optimizing culture conditions: taking glucose as a unique carbon source, wherein the concentration of the glucose is 15g / L; ammonium chloride is used as a sole nitrogen source, and the concentration of the ammonium chloride is 0.082 g / L; the culture temperature is 30 DEG C; the initial pH is 5; the phosphorus solubilizing amount of the phosphorus solubilizing fungi is 858.31 mg / L.

Description

Technical Field

[0001] This invention belongs to the fields of environmental and agricultural biotechnology, and specifically relates to the method for obtaining and optimizing a phosphate-solubilizing fungus. Background Technology

[0002] Phosphorus in soil is one of the essential nutrients for plant growth and development. However, most phosphorus in soil exists in the form of insoluble inorganic or organic phosphorus, which is difficult for plants to directly absorb and utilize, leading to problems such as low phosphate fertilizer utilization, resource waste, and environmental pollution. Microbial phosphate solubilization refers to the process of converting insoluble phosphorus into soluble phosphorus through microbial metabolic activities, and is an important way to improve the bioavailability of phosphorus in soil. Among them, phosphate-solubilizing fungi have significant application potential in sustainable agricultural production due to their strong environmental adaptability and phosphate-solubilizing ability.

[0003] Currently, various phosphate-solubilizing fungi have been isolated and reported, but the following problems still exist: First, most strains have limited phosphate-solubilizing efficiency, resulting in unstable effects in practical applications; second, the strains are mostly sourced from conventional farmland or forest land, with insufficient exploration of phosphate-solubilizing fungi resources specific to special habitats (such as grasslands and deserts); and third, the optimization of culture conditions for existing strains is not systematic enough, limiting their potential for large-scale application. Therefore, isolating highly efficient and stable phosphate-solubilizing fungi from special ecological environments and systematically optimizing their culture conditions is of great significance for developing efficient microbial phosphate fertilizers and promoting green agricultural development. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for obtaining and optimizing a phosphate-solubilizing fungus. This invention provides a *Penicillium fibrillans* strain isolated from soil in Xilingol League, Inner Mongolia. Penicillium steckii QF1 was developed, and its culture conditions were systematically optimized to achieve high phosphorus solubility.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for obtaining a phosphate-solubilizing fungus, comprising the following steps: S1. Collect soil samples from Xilingol League, Inner Mongolia; The coordinates of Xilingol League in Inner Mongolia are 43°57'48"N, 116°4'9"E.

[0006] S2. Soil samples were cultured in liquid and serially diluted under aseptic conditions. 100 μL of bacterial suspensions of different concentrations were spread on PVK solid medium supplemented with chloramphenicol and streptomycin. Single colonies with good growth, typical characteristics, and large and significant phosphate-solubilizing zones were selected and further purified by streaking on PVK solid medium to obtain pure cultures of the target strain. The cultures were preserved and identified to obtain phosphate-solubilizing fungi.

[0007] Preferably, the liquid culture temperature in S2 is 30°C, and the culture is carried out at 180 r / min for 30 min.

[0008] Preferably, the culture temperature on PVK solid medium in S2 is 30 ℃ and the culture time is 3 d to 10 d.

[0009] As a preferred option, the PVK liquid culture medium in S2 contains: glucose 10.0 g / L, Ca3(PO4)2 5.0 g / L, (NH4)2SO4 0.5 g / L, yeast extract 0.5 g / L, NaCl 0.3 g / L, MgSO4·7H2O 0.3 g / L, KCl 0.3 g / L, MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.03 g / L, streptomycin 50 mg / L, chloramphenicol 50 mg / L, pH 7.1~7.2; The PVK solid medium is composed of 18 g of agar added to each liter of PVK liquid medium.

[0010] Furthermore, the phosphate-solubilizing fungus is classified and named *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.

[0011] Secondly, the present invention provides an optimized method for the above-mentioned phosphate-solubilizing fungi, wherein the culture conditions for the phosphate-solubilizing fungi are as follows: Glucose is used as the sole carbon source, and the concentration of glucose is 15 g / L; ammonium chloride is used as the sole nitrogen source, and the concentration of ammonium chloride is 0.082 g / L. The incubation temperature is 30℃; The initial pH was 5.

[0012] Furthermore, the phosphorus-solubilizing capacity of the phosphate-solubilizing fungus is 858.31 mg / L.

[0013] It contains at least the following beneficial technical effects: This invention provides a strain of *Penicillium fibrillans* isolated from soil in Xilingol League, Inner Mongolia. Penicillium steckii The QF1 strain was developed, and its cultivation conditions were systematically optimized, resulting in highly efficient phosphorus solubility. Specific technical effects are as follows: This strain, derived from a typical grassland ecosystem, has previously been reported to possess phosphorus-solubilizing capabilities, enriching the germplasm resource bank of phosphorus-solubilizing fungi and providing a new material basis for subsequent genetic breeding and functional development. Through systematic optimization of key culture parameters such as carbon source, nitrogen source, temperature, and pH, the optimal combination was determined, and the strain achieved a phosphorus-solubilizing capacity of 858.31 mg / L, significantly higher than that of conventional phosphorus-solubilizing fungi, demonstrating excellent application potential. Attached Figure Description

[0014] 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.

[0015] Figure 2 The phosphorus solubility diagram of the target strain provided in Example 1 of this invention without condition optimization.

[0016] Figure 3 The phylogenetic tree of the target strain provided in Example 1 of this invention.

[0017] Figure 4 The Genbank accession number of the target strain provided in Example 1 of this invention.

[0018] Figure 5 A schematic diagram showing the determination results of the optimal growth temperature of the target strain provided in Example 2 of this invention.

[0019] Figure 6 A schematic diagram showing the determination results of the optimal pH of the target strain provided in Example 2 of this invention.

[0020] Figure 7 A schematic diagram showing the determination results of different carbon sources for the target strain provided in Example 2 of this invention.

[0021] Figure 8 A schematic diagram showing the determination results of different nitrogen sources for the target strain provided in Example 2 of this invention.

[0022] Figure 9 A schematic diagram showing the measurement results of the target strain on day 8 at different temperatures, pH, carbon sources, and nitrogen sources provided in Example 2 of this invention. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] Unless otherwise specified, "room temperature" and "normal temperature" in this invention refer to 25 ± 2 ℃.

[0029] Unless otherwise specified, all raw materials or instruments used in the following embodiments of the present invention are commercially available.

[0030] 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℃ and 180 r / min for 30 min. S3. Isolation and Culture: Under aseptic conditions, the soil suspension obtained from the shaker culture 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 medium supplemented with chloramphenicol and streptomycin. Three replicates were set up for each gradient. The cultures were incubated upside down at 30 ℃ for 3 to 10 days. Single colonies with good growth, typical characteristics, and large and prominent phosphate-solubilizing zones were picked with an inoculation loop and further purified by streaking on PVK solid medium to obtain pure cultures of the target strain, which were then preserved.

[0031] The isolation medium was PVK medium (g / L): glucose 10.0 g, Ca3(PO4)2 5.0 g, (NH4)2SO4 0.5 g, yeast extract 0.5 g, NaCl 0.3 g, MgSO4·7H2O 0.3 g, KCl 0.3 g, MnSO4·H2O 0.03 g, FeSO4·7H2O 0.03 g, streptomycin 50 mg, chloramphenicol 50 mg, pH 7.1~7.2.

[0032] PVK solid medium: Add 18 g of agar per liter of PVK liquid medium.

[0033] The preservation medium was PDB medium (1L formula): 200.0 g of peeled potatoes, cut into small pieces, added 1000 mL of water and boiled for 30 min. The mixture was filtered through double-layered gauze, and 20 g of glucose was added to the filtrate. The mixture was heated over low heat and stirred constantly with a glass rod. Distilled water was added to make up to 1000 mL, pH 5.

[0034] PDA medium: Add 18 g of agar per liter of PDA medium.

[0035] 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.

[0036] S4. Strain identification: Strain QF1 (e.g., obtained through steps S2 and S3) is then identified. Figure 1 (As shown), identified as the target strain; the phosphorus solubility of the strain was determined using the molybdenum-antimony colorimetric method (see...). Figure 2 After 9 days of culture, strain QF1 had a phosphorus solubility of 649.6 mg / L, indicating that strain QF1 has a strong phosphorus solubility.

[0037] The phosphorus solubility assay medium was NBRIP medium (g / L): 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, pH 7.2~7.4.

[0038] NBRIP solid medium: Add 18 g of agar per liter of NBRIP liquid medium.

[0039] The following is the identification of strain QF1: 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 ).

[0040] 18S rDNA analysis of B.QF1 strain Sequencing of QF1 yielded an 18S rDNA sequence of 571 bp, the DNA sequence of which is detailed in SEQ ID No. 1.

[0041] The 18S rDNA gene sequence of strain QF1 was analyzed for homology using NCBI's BLAST online analysis function, and a phylogenetic tree was constructed using MEGA software. The results showed that strain QF1 belongs to *Penicillium fimbriatum*. Currently, there are no literature reports on the phosphate-solubilizing ability of *Penicillium fimbriatum*, therefore, the identification 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.

[0042] Example 2 Optimization of culture conditions and determination of phosphorus solubility of strain Using NBRIP liquid medium as the basal medium, a 1% inoculum was added to the medium, with no inoculum serving as a blank control. The cultures were incubated at 28℃, 30℃, and 32℃ on a shaker at 180 r / min for 10 days, with three replicates. The cultures were centrifuged at 4℃ and 8000 r / min for 10 min, and the supernatant was used to determine the available phosphorus content using the molybdenum-antimony colorimetric method. The results are as follows: Figure 5 As shown, the optimal growth temperature for strain QF1 is 30℃. The phosphorus solubility of strain QF1 peaked on day 8. Comparing the phosphorus solubility of strain QF1 on day 8 revealed that the phosphorus solubility of strain QF1 at 30℃ was significantly higher than the other two treatments. The results are as follows: Figure 9 As shown. Therefore, the single-factor experiment results revealed that the optimal growth temperature for strain QF1 of the present invention is 30℃.

[0043] Using NBRIP liquid medium as the basal medium, a 1% inoculum was added to the medium, with an uninoculated control. Initial pH values ​​were set to 5, 6, and 7, respectively. The cultures were incubated at 30℃ and 180 r / min on a shaker for 10 days. Triple replicates were performed. The cultures were centrifuged at 4℃ and 8000 r / min for 10 min. The supernatant was collected, and the available phosphorus content was determined using the molybdenum-antimony colorimetric method. The results are shown below. Figure 6As shown, the three different pH treatments had no significant effect on the phosphorus solubility of strain QF1. The phosphorus solubility of strain QF1 peaked on day 8. Comparing the phosphorus solubility of strain QF1 on day 8, it was found that when the initial pH of the culture medium was 6, the phosphorus solubility of strain QF1 was slightly higher than the other two treatments, but the results were not statistically significant. Figure 9 As shown. Therefore, the optimal initial pH of the culture medium for strain QF1 could not be determined, nor could the initial pH of the culture medium be determined in relation to other factors. Therefore, the initial pH of the culture medium was added as an influencing factor in the orthogonal experiment to explore the interaction between multiple factors.

[0044] Using NBRIP liquid medium as the basal medium, glucose (10.0 g / L) in the NBRIP medium was replaced with equal amounts of sucrose (9.5 g / L) and starch (9.0 g / L) with the same carbon content. Inoculation was performed at a 1% inoculum volume, with an uninoculated control. The culture was incubated at 30℃ and 180 r / min on a shaker for 10 days. Triple replicates were performed. The culture was centrifuged at 4℃ and 8000 r / min for 10 min. The supernatant was collected, and the available phosphorus content was determined using the molybdenum-antimony colorimetric method. The results are shown below. Figure 7 As shown, the phosphate solubility of strain QF1 of this invention was best when glucose was the sole carbon source in the culture medium, reaching its peak on day 8. Comparison of phosphate solubility of strain QF1 on day 8 revealed that treatment with glucose as the sole carbon source was significantly higher than the other two treatments, as shown in the results below. Figure 9 As shown. Therefore, the single-factor experiment results showed that the strain QF1 of this invention had the best phosphate-solubilizing effect when glucose was the only carbon source in the culture medium.

[0045] Using NBRIP liquid medium as the basal medium, ammonium sulfate (0.1 g / L) in the NBRIP medium was replaced with equal amounts of ammonium chloride (0.082 g / L) and urea (0.054 g / L) with the same nitrogen content. Bacterial inoculum was added to the medium at a 1% inoculum level, with an uninoculated control. The medium was incubated at 30℃ and 180 r / min on a shaker for 10 days. Triple replicates were performed. The culture was centrifuged at 4℃ and 8000 r / min for 10 min. The supernatant was collected, and the available phosphorus content was determined using the molybdenum-antimony colorimetric method. The results are shown below. Figure 8 As shown, when ammonium sulfate was the sole nitrogen source in the culture medium, strain QF1 of this invention exhibited the best phosphorus solubility, reaching its peak on day 8. Comparison of phosphorus solubility of strain QF1 on day 8 revealed that treatment with ammonium sulfate as the sole nitrogen source significantly exceeded the other two treatments, as shown in the results below. Figure 9 As shown. Therefore, the single-factor experiment results showed that when the only nitrogen source in the culture medium was ammonium sulfate, the strain QF1 of this invention had the best phosphorus solubility.

[0046] Based on the single-factor experiment, appropriate influencing factors were selected for orthogonal experiment. A four-factor, three-level orthogonal experiment was adopted, using Table L9(3). 4 To investigate the optimal culture conditions for *Penicillium fibrillati*, the factors and levels of the orthogonal experiment are shown in Table 1. Liquid culture medium inoculated with *Penicillium fibrillati* was cultured under corresponding conditions. Every 24 hours, 5 mL of culture medium was collected to determine the phosphorus solubility. The initial phosphorus solubility on day 0 (before inoculation) was subtracted from the final result. The conditions and corresponding phosphorus solubility of the orthogonal experiment are shown in Table 2. K1, K2, and K3 represent the average values ​​of the same factor at different levels. Based on the results in Table 2 and further experimental verification, the order of influence on the phosphorus solubility of *Penicillium fibrillati* QF1 was determined to be A>D>C>B, i.e., glucose concentration>initial pH>nitrogen source>temperature. The optimal phosphorus solubility condition combination was A3B2C3D1, i.e., glucose 15 g / L, temperature 30°C, ammonium chloride as the nitrogen source, and initial pH 5. Since the orthogonal experiment did not include the optimal combination of *Penicillium fimbriatum*, a supplementary experiment was conducted to verify the practicality of the optimal combination obtained from the orthogonal experiment theory. Three parallel samples were set up with no inoculation as a blank control. The results showed that *Penicillium fimbriatum* QF1 reached its peak phosphorus solubility of 858.31 mg / L under the optimal combination conditions, which was the highest among all experimental groups, consistent with the theory.

[0047] Table 1. Factors and levels of orthogonal experiment Table 2 QF1 Orthogonal Experiment (L9(3) 4 ))result 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 method for obtaining a phosphate-solubilizing fungus, characterized in that, Includes the following steps: S1. Collect soil samples from Xilingol League, Inner Mongolia; The coordinates of Xilingol League in Inner Mongolia are 43°57'48"N, 116°4'9"E. S2. Soil samples were cultured in liquid and serially diluted under aseptic conditions. 100 μL of bacterial suspensions of different concentrations were spread on PVK solid medium supplemented with chloramphenicol and streptomycin. Single colonies with good growth, typical characteristics, and large and significant phosphate-solubilizing zones were selected and further purified by streaking on PVK solid medium to obtain pure cultures of the target strain. The cultures were preserved and identified to obtain phosphate-solubilizing fungi.

2. The acquisition method according to claim 1, characterized in that, The liquid culture temperature in S2 is 30 ℃, and the culture is carried out at 180 r / min for 30 min.

3. The acquisition method according to claim 1, characterized in that, The culture temperature of S2 on PVK solid medium is 30 ℃, and the culture time is 3 d to 10 d.

4. The acquisition method according to claim 1, characterized in that, The PVK medium in S2 consisted of: glucose 10.0 g / L, Ca3(PO4)2 5.0 g / L, (NH4)2SO4 0.5 g / L, yeast extract 0.5 g / L, NaCl 0.3 g / L, MgSO4·7H2O 0.3 g / L, KCl 0.3 g / L, MnSO4·H2O 0.03 g / L, FeSO4·7H2O 0.03 g / L, streptomycin 50 mg / L, chloramphenicol 50 mg / L, and pH 7.1–7.

2. The PVK solid medium is composed of 18 g of agar added to each liter of PVK liquid medium.

5. The method for obtaining according to claim 1, characterized in that, The phosphate-solubilizing fungus is classified and named Penicillium fimbriae ( ). Penicillium steckii It was deposited on January 8, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO: 41749.

6. The optimized method for the phosphate-solubilizing fungi according to claim 4, characterized in that, The culture conditions for the phosphate-solubilizing fungi are as follows: Glucose is used as the sole carbon source, and the concentration of glucose is 15 g / L; ammonium chloride is used as the sole nitrogen source, and the concentration of ammonium chloride is 0.082 g / L. The incubation temperature is 30℃; The initial pH was 5.

7. The optimization method according to claim 6, characterized in that, The phosphorus-solubilizing capacity of the phosphate-solubilizing fungus was 858.31 mg / L.

Images