Application of a melanin-producing Aureobasidium pullulans in improving saline soil and plant salt tolerance

Abstract

The application discloses application of melanin-producing Aureobasidium pullulans CNBG-PGPF-1 in improving saline soil and / or plant salt tolerance and / or promoting plant growth and belongs to the technical field of microorganisms.The strain CNBG-PGPF-1 has a promoting effect on seed germination and seedling growth of Medicago polymorpha under the condition of saline soil.The strain CNBG-PGPF-1 also enhances the salt stress tolerance of Medicago polymorpha by reducing the content of sodium ions, increasing the content of potassium ions, reducing the accumulation of hydrogen peroxide and increasing the content of proline.The melanin-producing Aureobasidium pullulans and the microbial agent thereof have a good application prospect in the improvement of saline soil and the production of crops in saline soil, and provide resource and technical support for further development of microbial agents or microbial manure suitable for the improvement of saline soil and the agricultural production in saline soil.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of a melanin-producing short-stemmed mold in improving saline soil and enhancing plant salt tolerance. Background Technology

[0002] Driven by multiple natural and anthropogenic factors, including climate change, seawater intrusion, and improper irrigation, soil salinization has become a global agricultural environmental problem, affecting a large amount of arable land worldwide. According to a 2024 report by the Food and Agriculture Organization of the United Nations (FAO), the area of ​​soil affected by salinization globally exceeds 1.381 billion hectares, accounting for approximately 10.7% of the global land area, seriously threatening global agricultural production. The high content of soluble salts and exchangeable sodium ions in saline soils leads to increased soil osmotic potential, which disrupts plant osmotic regulation mechanisms, triggering ion toxicity and oxidative stress, ultimately damaging various plant systems and affecting their growth and yield.

[0003] Currently, methods for improving saline soil mainly include agronomic measures, engineering measures, chemical amendments, and biological amendments. For example, by breeding and promoting salt-tolerant crop varieties, improving irrigation and drainage management, returning straw to the field, and applying chemical amendments, salt damage can be alleviated and soil structure improved to some extent. However, these methods still have certain limitations in terms of onset time, implementation cost, and applicability.

[0004] In recent years, the use of beneficial microorganisms to improve saline soils and promote crop growth has received widespread attention. Studies have shown that some microorganisms with plant growth-promoting effects can enhance plant tolerance to salt stress and promote growth and development through multiple pathways, including secreting secondary metabolites, promoting nutrient absorption, regulating ion balance, and inducing plant antioxidant pathways. Therefore, screening beneficial microorganisms that can stably colonize in saline soil environments and enhance crop salt tolerance and growth is of great significance for improving crop productivity in saline soils. Currently, the most widely used plant growth-promoting fungi are mostly concentrated in... Trichoderma genus, Fusarium No reports have been found on melanin-producing short-stemmed fungi, including those belonging to the genus and arbuscular mycorrhizal fungi, which have the effect of promoting crop growth and improving their tolerance to salt stress under saline soil conditions. Summary of the Invention

[0005] Purpose of the invention: The technical problem to be solved by this invention is to provide a melanin-producing short-stemmed mold (… Aureobasidium melanogenum Applications of CNBG-PGPF-1 in improving saline soil and / or plant salt tolerance and / or promoting plant growth.

[0006] Another technical problem to be solved by this invention is to provide a melanin-producing short-stemmed fungus ( Aureobasidium melanogenum Application of CNBG-PGPF-1 fermentation broth filtrate in improving saline soil and / or plant salt tolerance and / or promoting plant growth.

[0007] The final technical problem to be solved by this invention is to provide a melanin-producing short-stemmed fungus (… Aureobasidium melanogenum Application of CNBG-PGPF-1 formulations in improving saline soil and / or plant salt tolerance and / or promoting plant growth.

[0008] Technical solution: To solve the above technical problems, the present invention provides a melanin-producing short-stemmed mold (… Aureobasidium melanogenum Application of CNBG-PGPF-1 in improving saline soil and / or plant salt tolerance.

[0009] This invention also provides melanin-producing short-stemmed mold ( Aureobasidium melanogenum Application of CNBG-PGPF-1 fermentation broth filtrate in improving saline soil and / or plant salt tolerance and / or promoting plant growth.

[0010] This invention also provides a melanin-producing short-stem mold ( Aureobasidium melanogenum Application of CNBG-PGPF-1 formulations in improving saline soil and / or plant salt tolerance and / or promoting plant growth.

[0011] The preparation method of the fermentation broth filtrate includes the following steps: obtaining freshly cultured mycelial blocks of melanin-producing short-stem mold CNBG-PGPF-1 using a punch, placing them in PDB medium, culturing in the dark for 7-10 days, collecting the fermentation broth, filtering the fermentation broth with gauze, and then diluting it with sterile distilled water to obtain the fermentation broth filtrate.

[0012] The PDB culture medium comprises the following components: 3 g potato starch and 20 g glucose per 1000 mL of deionized water, sterilized by autoclaving.

[0013] The formulation includes single-ingredient or compound formulations, and the formulation type includes liquid formulations or solid formulations.

[0014] The application includes promoting plant growth in saline soil.

[0015] Specifically, the application includes: applying melanin-producing short-stemmed mold (… Aureobasidium melanogenum The fermentation broth filtrate of CNBG-PGPF-1 can be used as a base fertilizer or for seed soaking.

[0016] The concentration of spores of the melanin-producing short-stalked spore-forming fungus CNBG-PGPF-1 was 5 × 10⁻⁶. 4 Cells / mL ~2×105 per mL.

[0017] The plant mentioned includes one or both of alfalfa or tomato, and may also include other plants.

[0018] The application includes those that can tolerate salt concentrations of 0 to 0.6%.

[0019] Among them, the melanin-producing short-stemmed fungus CNBG-PGPF-1 can promote the germination of alfalfa seeds and / or the growth of seedlings. Furthermore, the pot experiment verified its promoting effect on the germination of alfalfa seeds and the growth of seedlings under saline soil conditions, as well as its enhancing effect on the salt stress tolerance of alfalfa, providing microbial products and technical solutions for improving saline soil and promoting crop growth.

[0020] Beneficial Effects: Compared with existing technologies, this invention has the following advantages: The strain CNBG-PGPF-1 of this invention promotes seed germination and seedling growth of alfalfa under saline soil conditions. The seed germination rate of the CNBG-PGPF-1 treatment group is more than double that of the control group. The aboveground fresh weight, underground fresh weight, and plant height of alfalfa seedlings are increased by 34.52%, 33.04%, and 13.83%, respectively, compared with the control group. CNBG-PGPF-1 also enhances the tolerance of alfalfa to salt stress by reducing sodium ion content, increasing potassium ion content, reducing hydrogen peroxide accumulation, and increasing proline content. The melanin-producing short-stemmed mold and its inoculant provided by this invention have good application prospects in saline soil improvement and saline soil crop production, providing resource and technical support for further development of inoculants or inoculants suitable for saline soil improvement and saline soil agricultural production. Attached Figure Description

[0021] Figure 1 The germination rate of alfalfa seeds in pot experiments in saline soil (C is the control group, F is the CNBG-PGPF-1 treatment group).

[0022] Figure 2 Photographs and growth indicators of alfalfa seedlings in pot experiments in saline soil (C is the control group, F is the CNBG-PGPF-1 treatment group).

[0023] Figure 3 The relative water content and relative electrolyte leakage data of alfalfa in pot experiments in saline soil are shown (C is the control group, and F is the CNBG-PGPF-1 treatment group).

[0024] Figure 4 The sodium and potassium ion content of alfalfa in pot experiments in saline soil (C is the control group, F is the CNBG-PGPF-1 treatment group).

[0025] Figure 5The hydrogen peroxide content of alfalfa in pot experiments in saline soil (C is the control group, F is the CNBG-PGPF-1 treatment group).

[0026] Figure 6 The proline content of alfalfa in pot experiments in saline soil (C is the control group, F is the CNBG-PGPF-1 treatment group).

[0027] Figure 7 The germination rate of tomato seeds was determined by a salt solution plate test (C is the control group, and F is the CNBG-PGPF-1 treatment group). Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings, embodiments, and experiments, but the present invention is not limited to the following technical solutions. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Those skilled in the art can implement the invention by referring to various commonly used reference books, scientific and technological documents, or related instructions and manuals prior to the filing date of this invention.

[0029] Example 1: Culture and fermentation broth preparation of melanin-producing short-skinned pupa CNBG-PGPF-1 Melanin-producing short-stemmed mold ( Aureobasidium melanogenum CNBG-PGPF-1, derived from patent application CN119193340A entitled "A plant endophytic fungus producing melanin-producing short-stalked fungus and its application", with accession number CGMCC No. 40635, was used for the daily activation, subculturing, and culture of this melanin-producing short-stalked fungus CNBG-PGPF-1 on potato dextrose agar (PDA) and cultured in the dark upside down in a constant temperature incubator at 25°C for 3-5 days to obtain fresh mycelia.

[0030] PDA formulation: 3 g potato extract powder, 20 g glucose, 15 g agar, add 1000 mL deionized water, autoclave at 121℃ for 20 min.

[0031] Preparation of fermentation broth filtrate of CNBG-PGPF-1: Freshly cultured CNBG-PGPF-1 (3-5 mycelial blocks) were obtained using a 5 mm punch, with each mycelial block having a volume of approximately 60 mm. 3 The spores were placed in 200 mL of PDB medium and incubated in the dark at 25°C and 160 rpm for 7 days. The fermentation broth was then collected. The broth was first filtered through gauze, and then diluted with sterile distilled water to a spore concentration of 2 × 10⁻⁶. 5 per mL.

[0032] PDB culture medium formula: 3 g potato extract powder, 20 g glucose, add 1000 mL deionized water, autoclave at 121℃ for 20 min.

[0033] Example 2: CNBG-PGPF-1 promotes alfalfa seed germination under saline soil conditions The tested plant material was alfalfa ( Medicago sativa Seeds of the L. variety "Mufeng" were purchased from Beijing Zhengdao Seed Industry Co., Ltd. The saline soil was collected from the coastal wetlands of Lianyungang City, Jiangsu Province, China. The main soil parameters of this saline soil were: pH 7.54, bulk density 1.42 g / cm³. 3 The salt content was 0.3%, and the contents of available nitrogen, available phosphorus, and available potassium were 26.5 mg / kg, 2.34 mg / kg, and 44.4 mg / kg, respectively. After collection, the sample was sieved through an 18-mesh sieve for later use. The treatment group used the CNBG-PGPF-1 fermentation broth filtrate prepared in Example 1.

[0034] The pot experiment included one control group (C) without fungal application and one treatment group (F) with 30 mL of CNBG-PGPF-1 fermentation broth filtrate applied as basal solution per pot. Each treatment had 10 replicates, for a total of 20 pots. Each pot contained 0.5 kg of saline soil and was sown with 12 alfalfa seeds. The pot experiments were managed under identical culture conditions. Seed germination was recorded 10 days after sowing.

[0035] Experimental results: After 10 days, the germination rate of alfalfa seeds in the control group was 34%, while the germination rate in the CNBG-PGPF-1 treatment group was 70%, representing an increase of more than 100%. Figure 1 ).

[0036] Example 3: CNBG-PGPF-1 promotes alfalfa seedling growth under saline soil conditions The plant materials, saline soil source, and pot experiment design and treatment methods were the same as in Example 2. After the alfalfa seeds germinated, they were cultured and thinned when the alfalfa plants grew to 10-15 cm, leaving 3 uniformly growing seedlings in each pot.

[0037] After three months of continuous cultivation from sowing, samples were collected and their growth indicators were measured.

[0038] Experimental results: After 3 months, compared with the control group, the alfalfa seedlings treated with CNBG-PGPF-1 fermentation broth filtrate had more stems and leaves, greener leaves, and more vigorous plants. Figure 2 ).like Figure 2 As shown, under the CNBG-PGPF-1 treatment, the aboveground fresh weight, underground fresh weight, and plant height of alfalfa seedlings were significantly higher than those of the control group, increasing by 34.52%, 33.04%, and 13.83%, respectively, indicating a significant growth-promoting effect.

[0039] Example 4: CNBG-PGPF-1 increases relative water content and reduces relative electrolyte leakage in alfalfa under saline soil conditions. After the pot experiments in Examples 1 and 2, the relative water content (RWC) of alfalfa leaves was determined by gravimetric method. Three leaves from the same part of the alfalfa plant were selected, and their fresh weight was recorded as FW. The leaves were then placed in a 50 mL centrifuge tube containing deionized water and soaked at room temperature for 24 h to allow the leaves to fully absorb water and reach saturation. After removing the leaves, the surface moisture was gently wiped off with filter paper, and the leaves were weighed again and recorded as saturated fresh weight (SFW). The leaves were then dried at 105℃ for 30 min, and then transferred to an 80℃ oven to dry to constant weight. After cooling to room temperature, the leaves were weighed and recorded as dry weight (DW). The relative water content of alfalfa leaves was calculated according to formula (1): (1) (1) In the formula: RWC This represents the relative water content of alfalfa leaves, expressed as % . FW The fresh weight of the leaves is in grams. SFW The saturated fresh weight of the leaves is expressed in grams. DW This represents the dry weight of the leaf blade, expressed in grams.

[0040] The relative electrolyte leakage (REL) of alfalfa leaves was determined using the conductivity method. Five leaves from the same part of an alfalfa plant were selected, cut into small sections, and placed in centrifuge tubes containing 20 mL of deionized water. The samples were soaked at room temperature for 24 h. The initial conductivity of the solution was measured using a conductivity meter and recorded as E1. The samples were then heated in a boiling water bath for 30 min to allow the cells to release all electrolytes. After the solution cooled to room temperature, the conductivity was measured again and recorded as E2. The relative electrolyte leakage of alfalfa leaves was calculated according to formula (2): (2) (2) Where: REL The relative electrolyte leakage rate of alfalfa leaves is expressed in % (%). E 1 represents the initial conductivity measured after immersion, in μS / cm; E 2 represents the final conductivity measured after boiling water bath treatment, in μS / cm.

[0041] Experimental results: After 3 months, the relative moisture content of alfalfa seedlings in the control group was 71%, while that in the CNBG-PGPF-1 treatment group was 80%, a significant increase of 12.68%. Figure 3 Meanwhile, the relative electrolyte leakage of alfalfa seedlings in the CNBG-PGPF-1 treatment group decreased by 8.57% ( Figure 3 ).

[0042] Example 5: CNBG-PGPF-1 reduces sodium ion content and increases potassium ion content in alfalfa under saline soil conditions. After the pot experiments in Examples 1 and 2, sodium ions (Na+) in alfalfa tissue were determined by inductively coupled plasma atomic emission spectrometry. + ) and potassium ions (K + Content. Weigh 0.1 g of uniformly ground alfalfa stems and leaves into a 50 mL centrifuge tube, add 15 mL of 1 mol / L hydrochloric acid solution, and extract on a shaker for 3 h to ensure complete leaching of ions from the sample. After extraction, centrifuge, collect the supernatant, and filter it to prepare the test solution.

[0043] The concentrations (denoted as C) of each ion in the solution were determined using inductively coupled plasma optical emission spectrometry (ICP-OES, iCAP 7400, Thermo Fisher Scientific, USA) and inductively coupled plasma mass spectrometry (ICP-MS, RQ, Thermo Fisher Scientific, USA). The sodium and potassium ion contents in alfalfa tissue were calculated according to formula (3): (3) (3) Where: C The concentration of ions in the solution to be tested is expressed in mg / L. V The volume is the solution volume, in mL. m The sample fresh weight is expressed in grams.

[0044] Experimental results: such as Figure 4 As shown, the sodium and potassium ion contents of alfalfa seedlings treated with CNBG-PGPF-1 changed significantly compared with the control group, decreasing by 20.16% and increasing by 37.86%, respectively, indicating that CNBG-PGPF-1 can alleviate sodium ion toxicity caused by salt stress.

[0045] Example 6: CNBG-PGPF-1 reduces hydrogen peroxide accumulation in alfalfa under saline soil conditions After the pot experiments in Examples 1 and 2, the hydrogen peroxide (H2O2) content in alfalfa tissue was determined by colorimetry. The reagents used were provided by the Hydrogen Peroxide (H2O2) Content Detection Kit - Colorimetric Method (D799773-0050) purchased from Sangon Biotech (Shanghai) Co., Ltd. Approximately 0.1 g of alfalfa stem and leaf samples were weighed, and 1 mL of H2O2 reagent was added. The mixture was thoroughly homogenized under ice bath conditions, and then centrifuged at 4℃ and 8000×g for 10 min. All supernatant was collected and placed on ice for later use.

[0046] Set up separate test tubes, standard tubes, and blank tubes. Add 1000 μL of the supernatant to the test tube, 1000 μL of 1 μmol / mL H₂O₂ standard to the standard tube, and 1000 μL of H₂O₂ reagent one to the blank tube. Then, add 100 μL of H₂O₂ reagent two and 200 μL of H₂O₂ reagent three to each reaction tube sequentially, mix well, and centrifuge at 4000×g for 10 min at room temperature. Discard the supernatant and retain the precipitate. Add 1000 μL of H₂O₂ reagent four to the precipitate, shake thoroughly to completely dissolve the precipitate, let stand at room temperature for 5 min, transfer to a cuvette, zero the instrument with distilled water, and measure the absorbance of each reaction tube at 415 nm. Record the absorbance as follows: A 测定管 , A 标准管 and A 空白管 The hydrogen peroxide content in alfalfa tissue is calculated according to formula (4): (4) (4) Where: H 2 O 2 The hydrogen peroxide content in the sample is expressed in μmol / g FW. Δ A 测定 To measure the difference in absorbance between the test tube and the blank tube, i.e., Δ A 测定 = A 测定管 - A 空白管 ; Δ A 标准 The difference in absorbance between the standard tube and the blank tube is Δ. A 标准 = A 标准管 - A 空白管 ; WThe sample fresh weight is expressed in grams.

[0047] Experimental results: After 3 months, the hydrogen peroxide content of alfalfa seedlings in the control group was 4.18 μmol / g FW, while that in the CNBG-PGPF-1 treatment group was 3.12 μmol / g FW, a significant reduction of 25.36%. Figure 5 ).

[0048] Example 7: CNBG-PGPF-1 increases proline content in alfalfa under saline soil conditions After the pot experiments in Examples 1 and 2, the free proline content in alfalfa tissue was determined by colorimetry. 0.1 g of uniformly ground alfalfa sample was weighed into a centrifuge tube, 5 mL of 3% sulfosalicylic acid solution was added, and the sample was extracted in a boiling water bath for 10 min. After cooling, the sample was filtered, and the filtrate was collected for later use. 2 mL of the filtrate was transferred to a test tube, and 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin solution were added sequentially. After mixing, the sample was placed in a boiling water bath for 30 min. After the reaction, the sample was immediately cooled to room temperature, and 4 mL of toluene was added to the test tube. The mixture was shaken for 30 s to extract the red compound generated by the reaction. After standing and separating the layers, the upper toluene phase was collected. Using toluene as a blank, the absorbance was measured at a wavelength of 520 nm. The proline concentration in the sample solution was calculated according to the proline standard curve, and the free proline content in the sample was calculated according to the method reported by Bates et al. The proline content in alfalfa tissue was calculated according to formula (5): (5) (5) In the formula: Proline The content of proline in the sample is expressed in μmol / g FW. C The concentration of proline is calculated from the standard curve, in μg / mL; V This represents the volume of toluene extraction, in mL. F The extraction volume correction factor is the ratio of the total volume of the extract to the volume of the reaction sample. 115.5 is the molecular weight of proline, in μg / μmol; W The sample fresh weight is expressed in grams.

[0049] Experimental results: After 3 months, compared with the control group, alfalfa seedlings treated with CNBG-PGPF-1 fermentation broth accumulated more proline, with a significantly increased content of 38.90%. Figure 6 ).

[0050] Example 8: CNBG-PGPF-1 promotes tomato seed germination under saline solution conditions Tomato (variety: large red tomato) seeds were purchased from Jiangsu Jiangshu Seedling Technology Co., Ltd. After surface disinfection, the seeds were divided into two groups: one group was treated with sterile distilled water, and the other group was treated with the CNBG-PGPF-1 fermentation broth filtrate prepared in Example 1. The seeds were completely immersed in either distilled water or fermentation broth filtrate at room temperature for 2 hours, then removed and drained for later use.

[0051] Tomato seeds were evenly placed in disposable petri dishes (9 cm in diameter) lined with two layers of qualitative filter paper. Each treatment group had 10 replicates, with 15 seeds in each dish. 3 mL of 0.6% NaCl solution was added, and the germination test was conducted in an artificial climate incubator at 25°C and 70% relative humidity. Seed germination was recorded after 8 days.

[0052] Experimental results: After 8 days, the germination rate of tomato seeds in the control group was 64%, while the germination rate in the CNBG-PGPF-1 treatment group was 83%, a significant increase of 29.69%. Figure 7 ).

Claims

1. Melanin-producing short-stalked mold ( Aureobasidium melanogenum Application of CNBG-PGPF-1 in improving saline soil and / or plant salt tolerance and / or promoting plant growth.

2. Melanin-producing short-stalked mold ( Aureobasidium melanogenum The fermentation broth filtrate of CNBG-PGPF-1 is preferably used in improving saline soil and / or plant salt tolerance and / or promoting plant growth. The preparation method of the fermentation broth filtrate is as follows: freshly cultured mycelial blocks of melanin-producing short-stem mold CNBG-PGPF-1 are obtained using a punch, placed in PDB medium, and cultured in the dark for 7-10 days. The fermentation broth is then collected, filtered through gauze, and then diluted with sterile distilled water to obtain the fermentation broth filtrate.

3. Contains melanin-producing short-stemmed fungus ( Aureobasidium melanogenum Application of CNBG-PGPF-1 formulations in improving saline soil and / or plant salt tolerance and / or promoting plant growth.

4. The application according to claim 3, characterized in that, The formulation includes single-ingredient or compound formulations, and the formulation type includes liquid formulations or solid formulations.

5. The application according to any one of claims 1 to 4, characterized in that, The applications include promoting plant growth in saline soils.

6. The application according to claim 5, characterized in that, The specific application includes: applying melanin-producing short-stemmed mold (… Aureobasidium melanogenum The fermentation broth filtrate of CNBG-PGPF-1 can be used as a base fertilizer or for seed soaking.

7. The application according to claim 5, characterized in that, The spore concentration of the melanin-producing short-spore fungus CNBG-PGPF-1 was 5 × 10⁻⁶. 4 Cells / mL ~2×10 5 per mL.

8. The application according to claim 1 or 5, characterized in that, The plant includes one or both of alfalfa and tomato.

9. The application according to claim 1 or 5, characterized in that, The applications include those that can tolerate salt concentrations of 0 to 0.6%.

10. The application according to claim 5, characterized in that, The melanin-producing short-stemmed mold CNBG-PGPF-1 can promote alfalfa seed germination and / or seedling growth.

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