Plant endophytic fungus melanin-producing aureobasidium and application thereof

Abstract

The invention discloses plant endophytic fungus melanin-producing aureobasidium and application thereof. The strain can effectively promote the growth of arabidopsis thaliana and tomatoes. After co-culture with the strain, the fresh weights of arabidopsis thaliana and tomato seedlings are respectively increased by 259.18% and 182.55% compared with those of a control group; and compared with a control group, the leaf area of the arabidopsis thaliana seedling is increased by 154.08%. After fermentation liquor filtrate of the strain is applied, in a potted plant growth promoting test, the fresh weight of the overground part of a tomato seedling is increased by 69.15% compared with a control group, the dry weight of the overground part is increased by 75.95%, the fresh weight of the underground part is increased by 80.00%, the dry weight of the underground part is increased by 120.01%, and the root length of the tomato seedling is increased by 66.98% compared with the control group. By applying the strain and the fermentation liquor filtrate thereof provided by the invention, a remarkable growth promoting effect can be exerted, and the strain has a wide development and utilization prospect in the field of agricultural microbial fertilizers and is worthy of being vigorously popularized.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a plant endophytic fungus, Acidis melanoderma pullulans, and application thereof. Background Art

[0002] Endophytic fungi are a type of fungi that colonize the internal tissues of plants. They can participate in the entire life cycle of the host plant and do not cause obvious harm to the host plant. Endophytic fungi are diverse and widely distributed in different plant tissues. They are widely used in agricultural and industrial production. In natural ecosystems, almost all living plants have endophytic fungi, which can be isolated from surface-sterilized plant tissues.

[0003] Endophytic fungi can establish a mutualistic symbiotic relationship with most plants in nature. Endophytic fungi can promote plant growth, enhance plant tolerance to abiotic and biotic stresses, and enhance defense responses, thereby having a beneficial effect on plants, while plants provide habitats and nutrients for endophytic fungi. By utilizing the plant-endophytic fungi symbiotic relationship, endophytic fungi can promote plant growth and promote the accumulation of secondary metabolites. Therefore, the isolation and use of endophytic fungi that have the ability to promote crop growth can help reduce the use of chemical fertilizers in agricultural ecosystems.

[0004] The fungus Aureobasidium melanogenum is one of the four variants of Aureobasidium pullulans. It has relatively more secondary metabolites, such as pullulan, polymalic acid and melanin. It has strong environmental adaptability and stress resistance. As a biocontrol fungus, it can effectively antagonize plant pathogenic fungi and bacteria. However, there are no reports on whether it can promote plant growth. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to isolate a plant endophytic fungus Aureobasidium melanogenum that can promote plant growth from the root system of thin-shelled pecan, and to study its effect on plant growth promotion, which provides a basis for the subsequent in-depth development of microbial fertilizers and the reduction of the use of chemical fertilizers.

[0006] The technical problem that the present invention also aims to solve is to provide a microbial preparation capable of promoting plant growth and a preparation method thereof.

[0007] The final technical problem to be solved by the present invention is to provide the application of endophytic fungus CNBG-PGPF-1 and microbial preparations in promoting plant growth.

[0008] Technical solution: In order to solve the above technical problems, the present invention provides a strain of Aureobasidium melanogenum CNBG-PGPF-1, which was deposited in the General Microbiological Center of China National Microbiological Culture Collection Committee on May 22, 2023, with a deposit number of CGMCC No.40635 and a deposit address of Beijing, China.

[0009] The present invention also includes a microbial preparation, which contains the Aureobasidium melanogenum CNBG-PGPF-1 or its fermentation broth filtrate.

[0010] Wherein, the preparation includes a single preparation or a compound preparation, and the preparation type includes a liquid preparation or a solid preparation.

[0011] The method for preparing the fermentation broth filtrate comprises the following steps: using a puncher to obtain a mycelium block of freshly cultured Aureobasidium melanogenum CNBG-PGPF-1, placing it in a PDB culture medium, collecting the fermentation broth after dark culture for 7-10 days, filtering the fermentation broth with gauze, and then diluting it with sterile distilled water to obtain a fermentation broth filtrate.

[0012] The PDB culture medium comprises the following components: 3 g potato extract powder and 20 g glucose per 1000 mL of deionized water, and is sterilized by high pressure steam.

[0013] The present invention also includes the application of the Aureobasidium melanogenum CNBG-PGPF-1 and the microbial preparation in promoting plant growth.

[0014] The concentration of Aureobasidium melanogenum CNBG-PGPF-1 spores in the microbial preparation is 5×10 4 / mL~2×10 5 Pieces / mL.

[0015] The plants include but are not limited to one or two of Arabidopsis and tomato, and are also suitable for promoting the growth of other plants.

[0016] The application specifically includes: co-culturing plants and Aureobasidium melanogenum CNBG-PGPF-1 on a solid culture medium.

[0017] Wherein, the solid culture medium includes but is not limited to MS culture medium. Preferably, the solid culture medium is 1 / 2MS solid culture medium.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following advantages: the strain of the present invention can effectively promote the growth of Arabidopsis and tomato; after co-cultivation with the strain, the fresh weight of Arabidopsis and tomato seedlings increased by 259.18% and 182.55% respectively compared with the control group; the leaf area of ​​Arabidopsis seedlings increased by 154.08% compared with the control group; after applying the fermentation filtrate of the strain, the fresh weight of the aboveground part of the tomato seedlings in the potted growth promotion test increased by 69.15% compared with the control group, the dry weight of the aboveground part increased by 75.95%, the fresh weight of the underground part increased by 80.00%, the dry weight of the underground part increased by 120.01%, and the root length of the tomato seedlings increased by 66.98% compared with the control group. The strain provided by the present invention and its fermentation filtrate are used for application, which can exert a significant growth-promoting effect and has broad development and utilization prospects in the field of agricultural microbial fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The colony morphology of the CNBG-PGPF-1 strain on PDA medium, the left side of the picture is the front view, and the right side is the back view.

[0020] Figure 2 This is the hyphae morphology of CNBG-PGPF-1 strain on PDA medium.

[0021] Figure 3 The spore morphology of CNBG-PGPF-1 strain on PDA medium.

[0022] Figure 4 This is the phylogenetic tree of the CNBG-PGPF-1 strain.

[0023] Figure 5 This is a growth-promoting effect diagram of the CNBG-PGPF-1 strain co-cultivated with Arabidopsis thaliana in 1 / 2MS medium. The upper column in the figure is the front view, and the lower column is the back view (Control is the control group, and CNBG-PGPF-1 is the treatment group).

[0024] Figure 6 The data show the growth-promoting effect of the CNBG-PGPF-1 strain co-cultivated with Arabidopsis thaliana in 1 / 2MS medium. The left side of the figure shows the fresh weight data, and the right side shows the leaf area data (Control is the control group, and CNBG-PGPF-1 is the treatment group).

[0025] Figure 7This is a diagram showing the growth-promoting effect of tomato after co-cultivation with the CNBG-PGPF-1 strain (Control is the control group, CNBG-PGPF-1 is the treatment group).

[0026] Figure 8 The data on the growth-promoting effect of tomato after co-cultivation with the CNBG-PGPF-1 strain are shown in the figure. The left side shows the fresh weight data, and the right side shows the hypocotyl length data (Control is the control group, and CNBG-PGPF-1 is the treatment group).

[0027] Fig. 9 This is the colonization of the CNBG-PGPF-1 strain on the tomato root system (Control is the control group, CNBG-PGPF-1 is the treatment group).

[0028] Fig.10 This is the effect of the CNBG-PGPF-1 strain on the biomass of tomato seedlings in a potted growth promotion experiment. The upper left figure shows the fresh weight data of the aboveground part, the upper right shows the dry weight data of the aboveground part, the lower left shows the fresh weight data of the underground part, and the lower right shows the dry weight data of the underground part (Control is the control group, CNBG-PGPF-1 10%, CNBG-PGPF-1 20%, and CNBG-PGPF-1 50% are treatment groups).

[0029] Fig.11 This is the effect of the CNBG-PGPF-1 strain potted growth promotion experiment on the growth indicators of tomato seedlings. The upper left part of the figure is the plant height data of the aboveground part, the upper right part is the root length data of the underground part, the lower left part is the SPAD data, and the lower right part is the leaf number data (Control is the control group, CNBG-PGPF-1 10%, CNBG-PGPF-1 20%, CNBG-PGPF-150% are the treatment groups). DETAILED DESCRIPTION

[0030] Below in conjunction with accompanying drawings and Examples and test, the present invention is further described in detail, but the present invention is not limited to the following technical scheme. Unless otherwise specified, the reagent, method and equipment adopted in the present invention are conventional reagents, methods and equipment in the art. Those of ordinary skill in the art can implement with reference to various commonly used reference books, scientific and technological literature or relevant specification sheets, manuals etc. before the present invention's application date.

[0031] Example 1 Isolation, purification and morphological observation of endophytic fungus CNBG-PGPF-1

[0032] The roots of healthy plants were collected at the thin-shell walnut planting base (Jurong, Jiangsu), and the root surface was washed with tap water. The samples were evenly cut into pieces and placed on filter paper for later use. First, the washed samples were placed in a 10mL centrifuge tube and washed with sterile water repeatedly by blowing and shaking; secondly, 75% ethanol solution was added for disinfection, and the samples were washed with repeated blowing and shaking, and the supernatant was removed after natural sedimentation; sterile water was taken again and the ethanol was washed by repeated blowing and shaking, and the ethanol was washed, and the mixture was repeated 3 times; then 10% sodium hypochlorite solution was added for disinfection, and the samples were washed with repeated blowing and shaking, and the supernatant was removed after natural sedimentation; finally, sterile water was taken and the sodium hypochlorite was washed by repeated blowing and shaking, and the mixture was repeated 3 times.

[0033] After the sample is disinfected, it is placed in a clean bench, cut open with a sterilized knife, placed on a fresh potato dextrose agar (PDA) plate, and cultured in the dark at 25°C. After the mycelium grows, according to the color, texture, and growth rate of the colonies, a sterile inoculation needle is used to pick up a single colony of fresh mycelium blocks and inoculate them into a new PDA plate, and then cultured in the dark in a 25°C constant temperature incubator for 3 to 5 days. After multiple subcultures, the pure strain CNBG-PGPF-1 was obtained, and the purified colonies were inoculated into slant cultures and stored at 4°C. In order to observe the colony, mycelium, and spore morphology of the CNBG-PGPF-1 strain, PDA medium was selected for culture.

[0034] PDA formula: 3 g potato powder, 20 g glucose, 15 g agar, add 1000 mL deionized water, and sterilize at 121°C high pressure steam for 20 min.

[0035] Experimental results: After 5 days of culture on PDA medium, the mycelium of CNBG-PGPF-1 was gray-green and flocculent, and the edges of the mycelium were dark green ( Figure 1 The hyphae of this strain are more branched and nearly transparent ( Figure 2 The spores of the strain are transparent, smooth-edged, and spindle-shaped or oval ( Figure 3 ), and the spore production was high, exceeding 10 / μL.

[0036] Example 2 Molecular Biological Identification of Endophytic Fungus CNBG-PGPF-1

[0037] Use a 5mm hole puncher to obtain the fungi cultured on the plate (3 mycelium blocks are sufficient), place them in a 2mL sterile tube containing 500μL 0.01M phosphate buffer (pH 7.2-7.4), and shake thoroughly to mix. Then add an appropriate amount of sterilized glass beads (dia. Glass beads, BioSpec) with a diameter of 0.5mm and 0.1mm (mix the two glass beads in equal proportions), and grind them with an oscillator at a speed of 6.5m / s for 1min. Finally, centrifuge at 12000rpm for 2min, and take the supernatant into a 1.5mL sterilized centrifuge tube for later use.

[0038] The crude DNA solution was used as a template, and ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') were used as upstream and downstream primers to perform PCR amplification on the ITS region fragment of the strain. The PCR amplification reaction system was 50 μL: 25 μL EasyTaq Mix, 2 μL forward primer ITS1, 2 μL reverse primer ITS4, 3 μL DNA template, and 17 μL sterile water. Amplification conditions: 94°C pre-denaturation for 5 min; 94°C denaturation for 30 s, 55°C annealing for 30 s, 72°C extension for 40 s, 36 cycles; 72°C extension for 10 min.

[0039] The PCR product was tested by 1% agarose gel electrophoresis and then sent to the company for sequencing. The sequencing results were compared with all ITS sequences in the GenBank database of NCBI using the Blast program, and the 10-15 species with the highest similarity were selected for phylogenetic tree analysis.

[0040] Experimental results: The sequencing results were spliced ​​to obtain a nearly full-length sequence of the ITS region of the strain CNBG-PGPF-1 (as shown in SEQ ID NO.1), which was 550bp in length. The ITS sequence of the strain was searched for homologous sequences in the GenBank database (NCBI), and the strain with the highest similarity was found using BLAST analysis. The results showed that the strain had a similarity of 98.14% with Aureobasidium melanogenum CBS.105.22. At the same time, a phylogenetic tree was constructed using MEGA 7 software and the Maximum Likelihood method, and it was found that the strain was clustered with multiple strains of Aureobasidium fungi, and was closest to Aureobasidium pullulans CBS 584.75 ( Figure 4 ), it can be determined that the strain is likely to be a variant of Aureobasidium pullulans, Aureobasidium melanogenum. CNBG-PGPF-1 was then deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of May 22, 2023, a deposit number of CGMCC No. 40635, and a classification name of Aureobasidium melanogenum.

[0041] Example 3 Co-cultivation experiment of CNBG-PGPF-1 strain and Arabidopsis thaliana in 1 / 2MS medium

[0042] (1) Disinfection of Arabidopsis seeds: 60-80 Arabidopsis seeds were placed in a 2 mL centrifuge tube for surface disinfection. First, 1 mL of sterile water was repeatedly blown and shaken to clean the seeds, and the supernatant was discarded after natural sedimentation. Secondly, 1 mL of 75% ethanol solution was repeatedly blown and shaken to disinfect the seeds for 2-3 min, and the supernatant was discarded after natural sedimentation. Then 1 mL of sterile water was repeatedly blown and shaken to wash away the ethanol, and the supernatant was discarded after natural sedimentation. Repeat this twice. Then 1 mL of 10% sodium hypochlorite solution was repeatedly blown and shaken to disinfect the seeds for 6-8 min, and the supernatant was discarded after natural sedimentation. Then 1 mL of sterile water was repeatedly blown and shaken to wash away the sodium hypochlorite, and the supernatant was discarded after natural sedimentation. Repeat this twice, and finally 1 mL of sterile water was drawn to resuspend the seeds.

[0043] (2) Spread the sterilized Arabidopsis seeds evenly on 1 / 2MS solid culture medium, discard the excess sterile water on the culture medium, blow dry it properly in a clean bench, seal it with a sealing film, and transfer it to a light culture room at 22°C, 16 h light / 8 h dark and culture it vertically until the seeds germinate.

[0044] (3) Sterile PDA blocks (left) with a diameter of 5 mm and a thickness of 3 mm and CNBG-PGPF-1 mycelium blocks (right) were placed on the left and right sides of the 1 / 2MS solid culture medium 0.9 cm away from the edge of the culture dish. The volumes of the PDA blocks and mycelium blocks were both close to 60 mm. 3 After the seeds germinate, select seedlings with uniform growth and place them evenly horizontally in a culture dish, 3 on each side, and seal them with sealing film. Transfer them to a light culture room and culture them vertically at 22°C, 16h light / 8h dark for 7-10 days. When there are obvious differences, collect the Arabidopsis seedlings and take pictures.

[0045] Experimental results: After 10 days, compared with the Arabidopsis seedlings in the control group, the Arabidopsis plants inoculated with CNBG-PGPF-1 were larger, had greener leaves, and had more developed root systems. In particular, the number of lateral roots of the Arabidopsis seedlings was significantly higher than that of the control group ( Figure 5 );like Figure 6 As shown, after inoculation with the CNBG-PGPF-1 strain, the fresh weight and leaf area of ​​Arabidopsis seedlings were significantly higher than those of the control group, which were increased by 259.18% and 154.08% respectively compared with the control group, and the growth-promoting effect was obvious.

[0046] Example 4 Co-cultivation experiment of CNBG-PGPF-1 strain and tomato in 1 / 2MS medium

[0047] (1) Tomato seeds are first disinfected: 60-80 tomato seeds are placed in a 15mL centrifuge tube for surface disinfection. First, 10mL of sterile water is repeatedly blown and shaken to clean the seeds, and the supernatant is removed after natural sedimentation. Secondly, 10mL of 75% ethanol solution is repeatedly blown and shaken to disinfect the seeds for 2-3 minutes, and the supernatant is removed after natural sedimentation. Then 10mL of sterile water is repeatedly blown and shaken to wash away the ethanol, and the supernatant is removed after natural sedimentation. Repeat this twice. Then 10mL of 10% sodium hypochlorite solution is repeatedly blown and shaken to disinfect the seeds for 6-8 minutes, and the supernatant is removed after natural sedimentation. Then 10mL of sterile water is repeatedly blown and shaken to wash away the sodium hypochlorite, and the supernatant is removed after natural sedimentation. After repeating this twice, 10mL of sterile water is sucked to resuspend the seeds.

[0048] (2) Spread the sterilized tomato seeds evenly on 1 / 2MS solid culture medium, discard the excess sterile water on the culture medium, blow dry it properly in a clean bench, seal it with a sealing film, mark it with a marker, and transfer it to a light culture room at 25°C, 12 h light / 12 h dark and culture it vertically until the tomato seeds turn white.

[0049] (3) Sterile PDA blocks (left) with a diameter of 5 mm and a thickness of 3 mm and CNBG-PGPF-1 mycelium blocks (right) were placed on the left and right sides of the 1 / 2MS solid culture medium 0.9 cm away from the edge of the culture dish. The volumes of the PDA blocks and mycelium blocks were both close to 60 mm. 3 After the seeds turn white, select tomato seeds with uniform growth and place them evenly horizontally in a culture dish, with 3 plants on each side. Seal the dish with sealing film and transfer it to a light culture room at 25°C, 12h light / 12h dark for 7-12 days. When there are obvious differences, collect the tomato seedlings, measure, photograph, and count the fresh weight and hypocotyl length.

[0050] Experimental results: After 12 days, Figure 7 and Figure 8 As shown, after inoculation with the CNBG-PGPF-1 strain, the root system of tomato seedlings was more developed, and the fresh weight and hypocotyl length were significantly higher than those of the control group, which were increased by 182.55% and 29.30% respectively compared with the control group, and the growth-promoting effect was obvious.

[0051] During the co-culture experiment, observations under an inverted microscope revealed that CNBG-PGPF-1 hyphae could invade the roots of tomato seedlings and extend further, with new hyphae extending out in a convex shape on the surface of the tomato roots ( Fig. 9 ), indicating that CNBG-PGPF-1 can successfully establish an interaction relationship with tomato.

[0052] Example 5 CNBG-PGPF-1 strain potted plant growth promotion test

[0053] (1) Tomato (variety: big red tomato) seedlings were raised in advance. One week later, seedlings of the same size were selected and transplanted into pots (10 cm × 10 cm) containing nutrient soil. One seedling was transplanted into one pot, and five pots were placed in each tray as five replicates. For each treatment of tomato plants, two trays with 10 replicates were set up.

[0054] (2) Use a 5 mm borer to obtain freshly cultured endophytic fungi (3-5 mycelium blocks), the volume of the mycelium block is about 60 mm 3 , put into 200mL PDB medium, culture in the dark at 25℃160rpm for 7 days, and collect the fermentation liquid. Take the fermentation liquid and filter it with gauze, then dilute it with sterile distilled water to 400mL as the fermentation liquid filtrate, the spore concentration is 1×10 5 Four treatments were set for tomatoes: sterile distilled water, 10% filtrate (spore concentration was 1×10 4 / mL), 20% filtrate (spore concentration is 2×10 4 / mL), 50% filtrate (spore concentration of 5×10 4 / mL), 40mL of solution was poured along the main root of each flower pot during treatment, and each tray required a total of 200mL of solution. The four treatments required 0mL, 400mL×10%=40mL, 400mL×20%=80mL, and 400mL×50%=200mL of fermentation liquid filtrate respectively.

[0055] PDB culture medium formula: 3 g potato powder, 20 g glucose, add 1000 mL deionized water, and sterilize with high-pressure steam at 121°C for 20 min.

[0056] (3) One week after transplanting tomatoes, root irrigation was carried out, and then treatment was carried out once every 10 days, and repeated three times. During this period, data such as plant height and relative chlorophyll content SPAD (Soil and Plant Analyzer Development) were measured; plant materials were collected when the growth difference between the treatments was visible to the naked eye, and data such as root length, fresh weight, and number of leaves were measured. After washing the plants, they were first sterilized at 105℃ for 30 minutes, and then placed in an oven at 80℃ to constant weight, and the dry weight data was measured.

[0057] Experimental results: Fig.10 As shown in the results, different concentrations of CNBG-PGPF-1 fermentation filtrate treatments all had good growth-promoting effects. Among them, 10% fermentation filtrate treatment significantly increased the aboveground fresh weight and aboveground dry weight of tomato plants, which were 69.15% and 75.95% higher than those of the control group, respectively. 20% fermentation filtrate treatment significantly increased the underground fresh weight of tomato plants, which was 80.00% higher than that of the control group; 50% fermentation filtrate treatment significantly increased the underground dry weight of tomato plants, which was 120.01% higher than that of the control group.

[0058] The results showed that the treatment with 10%, 20% and 50% CNBG-PGPF-1 fermentation filtrate significantly increased the root length of tomato plants, which was 63.55%, 66.98% and 50.16% higher than that of the control group, respectively. The treatment with the three concentrations of fermentation filtrate also significantly increased the SPAD value of tomato plants ( Fig.11 ).

Claims

1. A strain of Melanin-producing Aconitum Aureobasidium melanogenum ) CNBG-PGPF-1, characterized in that The melanin-producing short-stalked mold ( Aureobasidium melanogenum ) CNBG-PGPF-1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on May 22, 2023, with the deposit number: CGMCC No. 40635.

2. A microbial preparation, characterized in that: It contains the melanin-producing short-stalked mold ( Aureobasidium melanogenum ) CNBG-PGPF-1 or its fermentation broth filtrate.

3. The microbial preparation according to claim 2, characterized in that The preparation includes a single preparation or a compound preparation, and the preparation type includes a liquid preparation or a solid preparation.

4. The method for preparing the microbial preparation according to claim 2 or 3, characterized in that: The method for preparing the fermentation broth filtrate comprises the following steps: using a puncher to obtain freshly cultured black pigment-producing Acidis pulvinus ( Aureobasidium melanogenum ) The mycelium block of CNBG-PGPF-1 was placed in PDB medium and cultured in the dark for 7-10 days before the fermentation liquid was collected. The fermentation liquid was first filtered with gauze and then diluted with sterile distilled water to obtain the fermentation liquid filtrate.

5. The method for preparing the microbial preparation according to claim 4, characterized in that: The PDB culture medium comprises the following components: 3 g potato extract powder and 20 g glucose per 1000 mL of deionized water, and is sterilized by high pressure steam.

6. The melanin-producing short-stalked mold according to claim 1 ( Aureobasidium melanogenum ) Use of CNBG-PGPF-1 or the microbial preparation described in claim 2 or 3 in promoting plant growth.

7. The use according to claim 6, characterized in that: The microbial preparation contains the black pigment-producing Acidis sphaeroides ( Aureobasidium melanogenum ) The concentration of CNBG-PGPF-1 spores was 5×10 4 / mL~2×10 5 Pieces / mL.

8. The use according to claim 6, characterized in that: The plant comprises one or both of Arabidopsis thaliana and tomato.

9. The use according to claim 6, characterized in that: The application specifically includes: combining plants and melanin-producing Aconitum bacillus ( Aureobasidium melanogenum ) CNBG-PGPF-1 was co-cultivated with plants and endophytic fungi on solid medium.

10. The use according to claim 9, characterized in that: The solid culture medium includes MS medium.

Images