Phosphorus-solubilizing fungus, phosphorus-solubilizing bacterial agent and application thereof

Abstract

The invention relates to the field of environment and agricultural biotechnology, in particular to a phosphorus-solubilizing fungus, a phosphorus-solubilizing microbial inoculum and application thereof.The classification name of a phosphorus-solubilizing fungus strain XF2 is Penicillium deserticola, the strain XF2 is preserved in China General Microbiological Culture Collection Center (CGMCC) on January 9, 2025, and the preservation number is CGMCC NO: 41748. The bacterial colony characteristic of the strain XF2 is round, the edge is in a winding texture fluffy shape, the bacterial colony is yellow green or grey green, the surface is relatively smooth, and the bacterial colony can grow in a feather shape or a radial shape. Besides, after the strain XF2 is subjected to culture condition optimization, the phosphorus solubilizing amount can reach 874.3 mg / L. Therefore, the phosphorus solubilizing fungus has the advantage of high phosphorus solubilizing capacity, can be used for preparing a phosphorus solubilizing bacterial agent, has a very good application prospect, and particularly has a good application prospect in the aspects of dissolving phosphate ore and developing a phosphorus solubilizing fungus fertilizer.

Description

Technical Field

[0001] The invention relates to the fields of environment and agricultural biotechnology, and in particular to a phosphate-dissolving fungus, a phosphate-dissolving bacterial agent and applications thereof. Background Art

[0002] Phosphorus is one of the essential nutrients for plant growth and development. About 95% of phosphorus in the soil will be solidified by metal cations such as calcium, iron, and aluminum to form insoluble phosphates and accumulate, which can easily cause waste of phosphate resources and ecological and environmental problems such as soil compaction and environmental pollution. According to relevant reports, about 74% of cultivated land in my country is phosphorus-deficient, and the insufficient phosphorus content in the soil that can be directly absorbed and utilized by plants has become a major factor restricting crop growth and affecting agricultural output. At present, adding a large amount of chemical phosphate fertilizer is the main measure to supplement soil phosphorus and ensure grain production. However, due to the physical and chemical properties of the soil and the chemical properties of phosphorus, only 10% to 20% of the added phosphorus source can be absorbed and utilized by plants. In the long run, my country's already scarce high-grade phosphate resources face challenges. According to statistics, my country's total phosphate reserves are less than 6% of the world's total, while phosphate fertilizer production has increased nearly 100 times, making it the world's largest producer and consumer of phosphate fertilizers. Excessive reliance on phosphate fertilizers has increased agricultural production costs and increased the economic burden on farmers.

[0003] Soil microorganisms control key bio-geo-chemical processes in agricultural ecosystems and play a vital role in nutrient cycling and energy flow. Phosphate-solubilizing bacteria is a general term for microorganisms that have the ability to decompose phosphorus-containing compounds and promote the effectiveness of phosphorus, which can improve the utilization efficiency of phosphate fertilizers and phosphate rock. Phosphate-solubilizing bacteria are generally considered to be a high-quality alternative to chemical phosphate fertilizers. While promoting plant absorption of phosphorus, phosphate-solubilizing bacteria not only do not harm the agricultural ecological environment, but also have the potential to repair the soil environment polluted by traditional commercial fertilizers. There are many types of phosphate-solubilizing microorganisms, which can be divided into phosphate solubilizing bacteria (PSB), phosphate solubilizing fungi (PSF), phosphate solubilizing actinomyces (PSA) and cyanobacteria, among which phosphate-solubilizing bacteria account for 1% of the total phosphate-solubilizing microorganisms, and phosphate-solubilizing fungi account for 0.1% to 0.5%.

[0004] According to existing literature reports, phosphate-dissolving bacteria are widely distributed and strains with stronger abilities are not concentrated. Different types of bacterial resources may contain strains with higher phosphate-dissolving activity. From this perspective, the screening of wild strains with phosphate-dissolving ability is still of great significance. Studies have shown that fungi have much greater phosphate-dissolving performance than bacteria. In addition, phosphate-dissolving fungi also play an important role in improving plant response to biological and abiotic stresses such as drought, salinity, heavy metals, and pests and diseases. However, fungi are rarely used in actual agricultural production, so phosphate-dissolving fungi have gradually become a research hotspot in recent years.

[0005] In the prior art, there are few reports on phosphate-dissolving fungi, and the strain resource library of phosphate-dissolving fungi needs to be further enriched, and phosphate-dissolving fungal fertilizers need to be further developed. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the first object of the present invention is to provide a phosphate-dissolving fungus. The phosphate-dissolving fungus further enriches the genetic resources of wild phosphate-dissolving bacteria, expands the reserve library of phosphate-dissolving bacteria whole genome breeding, and further enriches the strain resource library of phosphate-dissolving fungi by obtaining living pure cultures of wild strains. The phosphate-dissolving fungus has a strong phosphate-dissolving ability, which is of great significance for the development of phosphate-dissolving bacterial fertilizers.

[0007] In order to overcome the shortcomings of the prior art, the second object of the present invention is to provide a phosphate-dissolving bacteria agent.

[0008] The third object of the present invention is to provide an application of phosphate-dissolving fungi.

[0009] In order to achieve the first object of the above invention, the technical solution adopted by the present invention is as follows:

[0010] The present invention provides a phosphate-dissolving fungus. The classification name of the phosphate-dissolving fungus strain XF2 is Penicillium desertorum (P. Desertorum). The strain XF2 was deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms on January 9, 2025, with a preservation number of CGMCC NO: 41748.

[0011] The colony of the strain XF2 is round, with a serpentine edge and a velvety texture, yellow-green or gray-green, a relatively smooth surface, and feather-like or radial growth on the colony. In addition, the strain XF2 can dissolve phosphate up to 874.3 mg / L after the culture conditions are optimized. Therefore, the phosphate-dissolving fungus of the present invention has the advantage of strong phosphate-dissolving ability and has a good application prospect, especially in the dissolution of phosphate ore and the development of phosphate-dissolving fungal fertilizer.

[0012] Further, the 16S rDNA gene sequence of the phosphate-dissolving fungus is shown in SEQ ID No. 1. Based on the gene sequencing results and the physiological and biochemical experimental results, it is determined that the phosphate-dissolving fungus belongs to Penicillium desertorum (P. Desertorum). The colony characteristics of the phosphate-dissolving fungus are round, with a serpentine edge and a velvety texture, yellow-green or gray-green in color, a relatively smooth surface, and feather-like or radial growth on the colony.

[0013] In order to achieve the second purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0014] The present invention provides a phosphate-dissolving bacterial agent, which contains the above-mentioned phosphate-dissolving fungi. The phosphate-dissolving fungi strain XF2 is prepared into the phosphate-dissolving bacterial agent, so as to better utilize the phosphate-dissolving ability of the phosphate-dissolving fungi strain XF2.

[0015] In order to achieve the third purpose of the above invention, the technical solution adopted by the present invention is as follows:

[0016] The present invention provides the use of the above-mentioned phosphate-dissolving bacteria agent in dissolving phosphate ore.

[0017] The present invention provides the use of the above-mentioned phosphate-dissolving bacteria agent as an agricultural microbial agent fertilizer.

[0018] The present invention provides application of the above-mentioned phosphate-dissolving bacteria agent in environmental restoration.

[0019] The present invention provides the use of the above-mentioned phosphate-dissolving fungi in dissolving phosphate ore.

[0020] The present invention provides the use of the above-mentioned phosphate-dissolving fungi as agricultural microbial fertilizer.

[0021] The present invention provides application of the above-mentioned phosphate-dissolving fungi in environmental restoration.

[0022] Among them, since the phosphate-dissolving fungus strain XF2 of the present invention has a strong phosphate-dissolving ability, the above-mentioned phosphate-dissolving bacteria or phosphate-dissolving fungi are applied to dissolving phosphate ore, the above-mentioned phosphate-dissolving bacteria or phosphate-dissolving fungi are applied to the preparation of agricultural microbial fertilizers, and the above-mentioned phosphate-dissolving bacteria or phosphate-dissolving fungi are applied to environmental remediation, all of which have good application prospects.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) A phosphate-dissolving fungus of the present invention, through morphological observation, 16SrDNA homologous sequence and phylogenetic tree analysis of the phosphate-dissolving fungus strain XF2, it is determined that the strain XF2 belongs to Penicillium desertorum. The strain XF2 enriches the genetic resources of wild phosphate-dissolving bacteria, expands the reserve library of phosphate-dissolving bacteria whole genome breeding, and further enriches the strain resource library of phosphate-dissolving fungi by obtaining live pure cultures of wild strains.

[0025] (2) Application of a phosphate-dissolving fungus of the present invention. Since the phosphate-dissolving fungus has a strong phosphate-dissolving ability, the strain XF2 can dissolve 874.3 mg / L of phosphate after the culture conditions are optimized. The phosphate-dissolving fungus also plays an important role in improving the ability of plants to cope with biological and non-biological stresses such as drought, salinity, heavy metals and pests and diseases. The phosphate-dissolving fungus or the phosphate-dissolving bacterial agent containing the phosphate-dissolving fungus can be used to dissolve phosphate ore, develop phosphate-dissolving bacterial fertilizers or environmental remediation, all of which have good application prospects and important significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 It is a colony morphology diagram of strain XF2 of the present invention.

[0028] Figure 2 This is a colony morphology diagram of the strain XF2 of the present invention after lactic acid phenol cotton blue staining.

[0029] Figure 3 It is a scanning electron micrograph of the strain XF2 of the present invention.

[0030] Figure 4 It is a schematic diagram of the change in the amount of solubilized phosphorus of the strain XF2 of the present invention during 9 days of cultivation.

[0031] Figure 5 It is a phylogenetic tree of strain XF2 of the present invention.

[0032] Figure 6 It is a screenshot showing the Genbank accession number of the strain XF2 of the present invention.

[0033] Figure 7 This is a graph showing the effect of different temperatures on the phosphorus solubilizing ability of strain XF2. DETAILED DESCRIPTION

[0034] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0035] The terms used in the embodiments of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. In the present invention, the singular forms "a", "said" and "the" used in the embodiments and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0036] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0037] The culture medium formula described in the following examples is as follows:

[0038] PVK medium (g / L): NaCl 0.2g, Ca 3 (PO 4 ) 2 5g, MgSO 4 0.1g, (NH 4 ) 2 SO 4 0.5g, glucose 15g, chloramphenicol 50mg, streptomycin 50mg, pH 6.8~7.0.

[0039] Solid culture medium of PVK: Add 18g to 20g of agar powder to every 1L of PVK culture medium.

[0040] PDA medium (g / L): 6 g potato extract, 20 g glucose, 18 g agar, natural pH.

[0041] PDA solid culture medium: Add 15g to 20g of agar powder to every 1L of PDA culture medium.

[0042] NBRIP medium (g / L): glucose 10 g, (NH 4 ) 2 SO 4 0.5g, NaCl 0.3g, MgSO 4 7H 2 O0.3g, FeSO 4 7H 2 O 0.03g, MnSO 4 ·2H 2 O 0.03g, CaCO 3 5g, KCl 0.3g, lecithin 0.2g, pH 7.2~7.4.

[0043] NBRIP solid culture medium: Add 18g to 20g of agar powder to every 1L of NBRIP culture medium.

[0044] Among them, PVK medium was used as the separation medium, PDA medium was used as the preservation medium, and NBRIP medium was used as the phosphate solubilization ability determination medium.

[0045] Example 1 Isolation and identification of strains

[0046] 1. Sample collection

[0047] Samples: Soil samples were collected from Xilin Gol League, Inner Mongolia (43°57'48"N, 116°4'9"E).

[0048] 2. Isolation and screening of strains

[0049] The separation and screening method adopts the enrichment culture method, which is as follows:

[0050] According to the soil fungus isolation procedure, the collected soil samples were enriched and cultured at 30°C and 180 r / min in a shaking incubator for 30 min; then, under sterile operation conditions, the soil suspension after enrichment culture in the shaking incubator was gradiently diluted in sequence to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 and 10 -6 Soil suspension of equal dilution gradient. 100 μL of bacterial solution of different dilution concentrations was applied to PVK medium supplemented with chloramphenicol and streptomycin, and 3 parallels were set for each gradient. Inverted culture was carried out at 30°C for 3 to 9 days. Then, a single colony with good growth, typical characteristics, and large and significant phosphate solubility zone was picked with an inoculation loop, and then streaked and purified on a solid separation medium (i.e., PVK solid medium) to obtain a pure culture of the target strain and preserve it.

[0051] A phosphate-dissolving fungus strain was successfully isolated from environmental samples using the above method and numbered XF2. The strain has a strong phosphate-dissolving ability, and the phosphate-dissolving capacity of strain XF2 can reach 874.3 mg / L after the culture conditions are optimized.

[0052] 2. Strain identification

[0053] The isolated strain XF2 with obvious phosphate solubility zone was Figure 1 The phosphate solubilizing ability of the strain was determined by the molybdenum antimony colorimetric method. Figure 4 As shown, after 9 days of cultivation, the amount of phosphorus solubilized by strain XF2 in the culture medium supernatant can reach 688.7 mg / L, indicating that strain XF2 has a strong phosphorus solubilizing ability.

[0054] Among them, in the process of determining the phosphorus solubilizing ability of the strain using the molybdenum antimony colorimetric method, the strain with the strongest phosphorus solubilizing ability was selected. After the purified phosphorus solubilizing bacteria were inoculated into the PDA liquid culture medium for culture, the bacterial solution was inoculated into the NBRIP culture medium at a 1% inoculation rate, and no inoculation of bacterial solution was set as a control, and 3 groups of parallel experiments were set up. The culture was carried out at 30°C and 180r / min in a shaker for 9 days, and samples were taken every 24 hours, and then centrifuged at 4°C and 8000r / min for 10 minutes, and then the supernatant was taken to determine the effective phosphorus content using the molybdenum antimony colorimetric method.

[0055] Among them, the colony characteristics of strain XF2 are round, with winding edges and velvety texture, yellow-green or gray-green color and relatively smooth surface, and feather-like or radial growth on the colony. Figure 1 and Figure 2 As shown, Figure 2 The colony morphology of strain XF2 was stained with lactic acid phenol cotton blue. In addition, the strain XF2 was observed using a scanning electron microscope, and its scanning electron microscope image is as follows Figure 3 shown.

[0056] 16S rDNA molecular biological identification:

[0057] The strain XF2 was sequenced to obtain the 16S rDNA gene sequence of the strain XF2, which is 557 in length. Please refer to the sequence table for its DNA sequence.

[0058] Among them, the blast online analysis function of NCBI was used to perform homology search on the 16S rDNA gene sequence of strain XF2, and the phylogenetic tree was constructed and the evolutionary relationship was analyzed using MEGA software.

[0059] Phylogenetic tree of 16S rDNA Figure 5 As shown, the results show that the strain XF2 isolated and purified by the present invention belongs to Penicillium desertorum (P. Desertorum). There is no literature report on the ability of Penicillium desertorum to dissolve phosphate, so the screening of strain XF2 enriches the genetic resources of wild phosphate-dissolving bacteria and expands the reserve library for whole genome breeding of phosphate-dissolving bacteria. The gene sequence of strain XF2 has been submitted to the Gen Bank database with accession number PQ756957, please see Figure 6 shown.

[0060] In addition, the strain XF2 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on January 9, 2025, with the deposit number CGMCC NO: 41748, and the deposit address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0061] Example 2 Optimization of culture conditions and detection of phosphate solubilization ability of strain XF2

[0062] Using NBRIP liquid medium as the basic medium, strain XF2 was inoculated with 1% bacterial solution in the medium, and no bacterial solution was used as the control. The medium was cultured at 25℃, 28℃ and 30℃ at 180r / min for 10 days, and three parallel cultures were set up. The amount of dissolved phosphorus was tested every day from 1d to 10d of culture. The test results are shown in the figure. Figure 7 shown.

[0063] Depend on Figure 7 It can be seen that the single factor test results found that the most suitable culture temperature for the desert Penicillium of the present invention is 30°C.

[0064] In addition, based on the above single factor test results, an orthogonal test was conducted on the culture conditions of strain XF2. The orthogonal test conditions and the corresponding phosphorus solubilization amount are shown in Table 1.

[0065] Table 1 Orthogonal test table of culture conditions of strain XF2

[0066]

[0067]

[0068] The results in Table 1 combined with further experimental verification determined that the optimal growth conditions of strain XF2 are: the initial pH of the NBRIP liquid culture medium is 5, the concentration of glucose is 10 g / L, the concentration of ammonium sulfate is 0.5 g / L, and the concentration of calcium phosphate is 7 g / L. Under the above optimal growth conditions, the strain XF2 can solubilize 874.3 mg / L of phosphorus, indicating that the phosphorus-dissolving fungus of the present invention has a strong phosphorus-dissolving ability.

[0069] Example 3

[0070] A phosphate-dissolving bacterial agent contains the phosphate-dissolving fungi of the present invention. The phosphate-dissolving fungi strain XF2 is prepared into the phosphate-dissolving bacterial agent, so as to better utilize the phosphate-dissolving ability of the phosphate-dissolving fungi strain XF2.

[0071] Example 4

[0072] An application of phosphate-dissolving fungi, wherein the phosphate-dissolving fungi of the present invention is applied to dissolving phosphate ore, or the phosphate-dissolving fungi of the present invention is applied to preparing agricultural microbial fertilizer, or the phosphate-dissolving fungi of the present invention is applied to environmental remediation. Since the phosphate-dissolving fungi strain XF2 of the present invention has a strong phosphate-dissolving ability, the phosphate-dissolving fungi of the present invention is applied to dissolving phosphate ore, preparing agricultural microbial fertilizer, or environmental remediation, all of which have good application prospects.

[0073] Example 5

[0074] An application of a phosphate-dissolving bacteria agent, wherein the phosphate-dissolving bacteria agent of the present invention is applied to dissolving phosphate ore, or the phosphate-dissolving bacteria agent of the present invention is applied to preparing agricultural microbial fertilizer, or the phosphate-dissolving bacteria agent of the present invention is applied to environmental remediation. Since the strain XF2 in the phosphate-dissolving bacteria agent of the present invention has a strong phosphate-dissolving ability, the phosphate-dissolving bacteria agent of the present invention is applied to dissolving phosphate ore, preparing agricultural microbial fertilizer, or environmental remediation, all of which have good application prospects.

[0075] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

[0076] .

Claims

1. A phosphate-dissolving fungus, characterized in that: The phosphate-solubilizing fungus strain XF2 is classified and named as Penicillium Desertorum (P. Desertorum). The strain XF2 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on January 9, 2025, with the deposit number CGMCC NO: 41748.

2. A phosphate-dissolving fungus according to claim 1, characterized in that: The 16SrDNA gene sequence of the phosphate-solubilizing fungus is shown as SEQ ID No.

1.

3. A phosphate-dissolving bacteria agent, characterized in that: Contains the phosphate-dissolving fungus according to claim 1 or 2.

4. Use of the phosphate-dissolving bacteria agent according to claim 3 in dissolving phosphate ore.

5. Use of the phosphate-dissolving bacteria agent according to claim 3 as an agricultural microbial fertilizer.

6. Use of the phosphate-dissolving bacteria agent according to claim 3 in environmental remediation.

7. Use of the phosphate-dissolving fungus according to claim 1 or 2 in dissolving phosphate ore.

8. Use of the phosphate-dissolving fungus according to claim 1 or 2 as an agricultural microbial fertilizer.

9. Use of the phosphate-dissolving fungi according to claim 1 or 2 in environmental remediation.

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