Phaeosphaeria nodorum c1-3 and application thereof

Abstract

This invention relates to a strain of *Pseudomonas verrucosum* C1-3 and its applications, belonging to the field of biotechnology. The *Pseudomonas verrucosum* (… Paraphaeosphaeria verruculosa The preservation number of C1-3 is: CCTCC NO: M 2026903. The verrucous pseudoglobulus C1-3 can be used to dissolve inorganic phosphorus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a strain of *Pseudomonas verrucosum* C1-3 and its applications. Background Technology

[0002] Phosphorus is an essential macronutrient for plant growth, participating in various physiological processes such as energy metabolism, nucleic acid synthesis, and membrane structure construction. While soil contains relatively abundant total phosphorus, most of it exists as insoluble phosphates, including calcium phosphate, aluminum phosphate, iron phosphate, and phosphate rock powder, which plants cannot directly absorb and utilize. Statistics show that approximately 74% of arable land in my country suffers from phosphorus deficiency or severe phosphorus deficiency, necessitating the extensive use of chemical phosphate fertilizers in agricultural production. However, the utilization rate of chemical phosphate fertilizers applied to the soil is only 10%–25% within the same season, with the vast majority fixed in the soil in forms unusable by plants. This results in significant waste of phosphate fertilizer resources, and excessive application of phosphate fertilizers also contributes to environmental problems such as eutrophication of water bodies.

[0003] Phosphate-solubilizing microorganisms (PSMs) can convert insoluble phosphates in soil into available phosphorus that can be absorbed and utilized by plants through mechanisms such as secretion of organic acids, acid production, and enzyme production. This is an important biological pathway for improving soil phosphorus nutrition. Existing research reports mainly include Bacillus species (…). Bacillus ), Pseudomonas spp. Pseudomonas ), Penicillium ( Penicillium Aspergillus ( ) Aspergillus However, these reported strains still suffer from problems such as low phosphorus solubilization efficiency, poor environmental adaptability, and limited functionality in practical applications, necessitating the discovery of novel, highly efficient phosphorus-solubilizing microbial resources.

[0004] Verrucous globosum ( Paraphaeosphaeria verruculosa *[unclear text - likely a typo]* is a filamentous fungus found in soil, plant debris, and other environmental environments. Related studies have shown that its fermentation products possess antifungal activity against plant pathogens. Furthermore, oxalic acid is considered one of the important metabolites released by the fungus in response to environmental stress. Currently, there are no published reports on [unclear text - likely related to oxalic acid]. Paraphaeosphaeria verruculosa It has phosphorus solubilization function. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention provides a strain of Pseudomonas verrucosum C1-3 and its application.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: The first aspect of the present invention provides a strain of *Pseudomonas verrucosum*, wherein *Pseudomonas verrucosum* ( Paraphaeosphaeria verruculosaThe accession number for C1-3 is CCTCC NO: M 2026903, and its classification name is: Paraphaeosphaeria verruculosa C1-3 was deposited on May 8, 2026 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, 430072, China.

[0007] A second aspect of the present invention provides a bacterial agent, wherein the active ingredient of the bacterial agent is *Pseudomonas verrucous* as described in the first aspect.

[0008] The third aspect of the present invention provides the use of *Pseudomonas verrucosum* of the first aspect and / or the bacterial agent described in the second aspect in the dissolution of inorganic phosphorus. Beneficial effects

[0009] The *Pseudomonas verrucosum* provided by this invention can be used to dissolve inorganic phosphorus. Attached Figure Description

[0010] Figure 1 This is a photograph of the C1-3 plate culture of *Pseudomonas verrucosum* in this invention. The left image shows the front of the plate, and the right image shows the back of the plate. Figure 2 This is the phylogenetic tree of *Pseudomonas verrucosum* C1-3 in this invention; Figure 3 This is a photograph of the first replicate of the three replicates used in the qualitative detection of the phosphate-solubilizing function of *Pseudomonas verrucous* C1-3 in this invention. Figure 4 This is a photograph of the second replicate of the three replicates for the qualitative detection of the phosphate-solubilizing function of *Pseudomonas verrucous* C1-3 in this invention. Figure 5 This is a physical image of the third replicate in the three replicates of the qualitative detection of the phosphate-solubilizing function of *Pseudomonas verrucous* C1-3 in this invention. Biological Preservation

[0011] The present invention provides *Pseudomonas verrucosum* (a type of bacteria). Paraphaeosphaeria verruculosa C1-3, categorized and named as follows: Paraphaeosphaeria verruculosa C1-3 was deposited at the China Center for Type Culture Collection (CCTCC) on May 8, 2026, with accession number CCTCC NO: M 2026903. The address of the collection center is Wuhan University, Wuhan, China, 430072, China. Detailed Implementation

[0012] The present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention. Example

[0013] Verrucous globosum ( Paraphaeosphaeria verruculosa Experimental steps for the separation, purification, and phosphorus solubilization of C1-3.

[0014] I. Preparation of Experimental Materials and Instruments 1.1 Test Sample Healthy shallot plants in the field during their growth period were sampled, and soil samples with roots tightly attached to the rhizosphere (0-2mm) were collected. The samples were then sealed in sterile self-sealing bags and stored at 4℃. Strains were isolated within 24 hours. Mixed rhizosphere soil samples were prepared by sampling from five points, avoiding diseased plants and areas heavily affected by fertilization.

[0015] 1.2 Culture medium formulation (all autoclaved at 121℃ for 20 min) 1.2.1 Used for targeted isolation of ascomycetes Paraphaeosphaeria verruculosa PDA selective culture medium (inhibits bacteria) 200 g of potatoes (peeled, diced, boiled in water, and the juice filtered out), 20 g of glucose, and 20 g of agar were added, and the volume was adjusted to 1000 mL with deionized water. After autoclaving, when the culture medium cooled to approximately 55 °C, chloramphenicol and streptomycin solutions filtered through a sterile membrane were added to achieve final concentrations of 50 mg / L and 100 mg / L, respectively, to inhibit bacteria and actinomycetes in the soil.

[0016] 1.2.2 Strain purification using PDA solid / liquid culture medium Antibiotic-free PDA medium is used for strain passage, morphological observation, and strain preservation.

[0017] 1.2.3 Inorganic phosphorus screening solid culture medium (NBRIP, phosphorus solubilization qualitative analysis) 10g glucose, 5g MgCl2•6H2O, 0.25g MgSO4·7H2O, 0.2g KCl, 0.1g (NH4)2SO4, 5g insoluble tricalcium phosphate Ca3(PO4)2, 20g agar, 1000mL deionized water, pH=7.0; the presence of a transparent phosphate-solubilizing zone around the colony indicates phosphate-solubilizing fungi.

[0018] 1.2.4 Sterile diluent 0.85% sterile saline, dispensed in 9 mL / test tube, for soil gradient dilution.

[0019] 1.3 Reagents and Consumables ITS Fungal DNA Extraction Kit, PCR Primers (ITS1 / ITS4), 75% ethanol, sterile culture dishes, centrifuge tubes, pipette tips, sterile filter paper, soil drill, and dissecting scissors.

[0020] 1.4 Experimental Apparatus Clean bench, vertical autoclave, constant temperature fungal incubator, PCR amplifier, stereomicroscope, optical microscope, electronic balance.

[0021] II. Pretreatment steps for scallion rhizosphere soil samples 1. Sample pretreatment: Take out the scallion roots in the laminar flow hood, gently shake off the loose soil, leaving 2mm of soil adhering to the root surface; use a sterile brush to collect the root soil, pass it through a 2mm sterile sieve to remove plant debris and gravel, and mix well for later use.

[0022] 2. Preparation of soil fungal suspension: Accurately weigh 10g of well-mixed rhizosphere soil and add it to a 90mL sterile Erlenmeyer flask containing physiological saline. Incubate at 28℃ and 180rpm for 30min to release soil-encapsulated fungal spores and hyphae. Allow to stand for 5min to allow large soil particles to settle, obtaining 10... -1 Soil mother liquor.

[0023] 3. Serial dilution: Perform serial dilution aseptically, preparing 10 batches sequentially. -2 10 -3 10 -4 10 -5 10 -6 Dilute the bacterial suspension. Paraphaeosphaeria verruculosa It is a low-abundance dark-colored fungus in the rhizosphere, preferably 10 -3 10 -4 10 -5 Gradient coating.

[0024] 3.1 Initial Separation of Flat Plate Coating 3.1.1 Sterilize with ultraviolet light in a clean bench for 30 minutes, prepare antibiotic PDA selective plates in advance, and let them cool and solidify for later use.

[0025] 3.1.2 Take 100 μL of bacterial suspension for each gradient and spread it evenly on a selective PDA plate. Perform 3 biological replicates for each gradient.

[0026] 3.1.3 Invert the plate and incubate at 25℃ in the dark for 7-10 days (the growth rate of this fungus is slower than that of common molds, so the incubation period should be extended).

[0027] 3.2 Initial screening of target strains (preliminary assessment of colony morphology) Paraphaeosphaeria verruculosa Standard colony characteristics (as shown in the attached document) Figure 1 (As shown): The colonies are flat, dense, and velvety in texture, grayish-white on the front and light brownish-brown on the back. The pigment is darker in the center of the colony, gradually fading towards the periphery, with no pigment diffusion. The hyphae are dark and produce brown conidia, distinguishing it from Penicillium, Aspergillus, and Fusarium. Select single colonies that match the morphological characteristics.

[0028] 3.3 Strand Purification 3.3.1 Select young hyphae from the edge of the target single colony, streak them in four consecutive zones on an antibiotic-free PDA plate for purification, and incubate at 25°C for 5 days.

[0029] 3.3.2 Repeat the streak purification process three times to completely remove soil-associated bacteria and obtain pure culture strains.

[0030] 3.3.3 The purified strain was stored on slant at 4℃ and then stored at -80℃ with 20% glycerol for long-term preservation.

[0031] 3.4 ITS Molecular Biological Identification (1) Scrape pure culture hyphae and extract fungal genomic DNA using a kit; (2) ITS1 / ITS4 primers were used to amplify the conserved fungal ITS sequence by PCR. (3) After sequencing, NCBI BLAST homology comparison showed a sequence similarity of ≥98.33%, and the phylogenetic tree was constructed as shown in the attached figure. Figure 2 As shown, the strain was identified as Paraphaeosphaeria verruculosa .

[0032] (4) ITS gene sequence (SEQ ID NO.1): >C1-3 .

[0033] III. Qualitative Detection of Phosphate Solubilization Function of Strains (Transparency Zone Method) 1. Activated strain: P. verruculosa was purified by transfer onto PDA plates, cultured at 25°C for 6 days, and sterile mycelial cakes with a diameter of 5 mm were prepared.

[0034] 2. Inoculation with mycelium: Invert the mycelium and inoculate it in the center of an NBRIP inorganic phosphorus solid plate, with 3 replicates per group.

[0035] 3. Constant temperature incubation: Incubate at 25℃ in the dark for 8 days.

[0036] 4. Index determination: The diameter of the colony (D) and the diameter of the outer transparent phosphate-solubilizing zone (d) were measured by the cross-cross method. The phosphate solubility index SI = d / D was calculated, and the results are shown in Table 1 below.

[0037] Table 1. Statistical table of phosphorus solubilization function of C1-3 *Pseudomonas verrucous*. 1 1.52 4.56 3.00 2 1.44 4.38 3.04 3 1.51 4.61 3.05 Average value 1.49 4.52 3.03 5. Result Interpretation: The appearance of a clear, colorless, and transparent hydrolysis zone on the plate proves that the strain has the ability to dissolve inorganic, sparingly soluble phosphorus.

[0038] The above description is merely a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the concept described herein through the above teachings or related technologies or knowledge. Modifications and variations made by those skilled in the art that do not depart from the spirit and scope of the present invention should be within the protection scope of the appended claims.

Claims

1. A strain of *Pseudomonas verrucous* C1-3, characterized in that: The verrucous coccidioides ( Paraphaeosphaeria verruculosa The accession number for C1-3 is CCTCC NO: M 2026903, and its classification name is: Paraphaeosphaeria verruculosa C1-3.

2. A bacterial agent, wherein the active ingredient of the bacterial agent is *Pseudomonas verrucosum* C1-3 as described in claim 1.

3. The use of *Pseudomonas verrucosum* C1-3 as described in claim 1 and / or the bacterial agent as described in claim 2 in the dissolution of inorganic phosphorus.

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