Plant endophytic fungus strain of aureobasidium melanogenum and application thereof

Abstract

Aureobasidium melanogenum CNBG-PGPF-1 deposited with China General Microbiological Culture Collection Center on May 22, 2023 under accession number CGMCC No. 40635. Microbial formulation comprising A.

Description

TECHNICAL FIELD The present invention belongs to the field of microbial technology, and relates to a plant endophytic fungus strain of Aureobasidium melanogenum and application thereof. BACKGROUND As a class of fungi that colonize internal tissues of plants, endophytic fungi participate in an entire life cycle of the host plant without causing obvious harm to the host plant. Endophytic fungi are diverse and widely distributed in various plant tissues, with broad application prospects in agricultural and industrial production. In natural ecosystems, endophytic fungi can be found in almost all living plants, and are usually isolated from surface-sterilised plant tissues. Endophytic fungi are capable of establishing mutualistic symbiotic relationships with the vast majority of plants, promoting plant growth, enhancing plant tolerance to both abiotic and biotic stresses, and strengthening plant defence responses, thereby having beneficial effects on the plants. In return, the plants provide habitats and nutrients for the endophytic fungi. By utilizing the symbiotic relationship with plants, endophytic fungi can promote the growth of plants and facilitate the accumulation of secondary metabolites. Therefore, the isolation and utilization of endophytic fungi with plant growth-promoting capabilities may contribute to reducing the use of chemical fertilizers in agricultural ecosystems. Aureobasidium melanogenum is one of the four variants of Aureobasidium pullulans. It is known to produce various secondary metabolites, such as pullulan, polymalic acid, and melanin. It has strong environmental adaptability and stress resistance. As a biocontrol agent, it can effectively antagonize phytopathogenic fungi and bacteria. However, there have been no reports on whether it can promote plant growth yet. SUMMARY Objectives of the present invention: One technical problem to be solved by the present invention is to isolate a plant growth-promoting endophytic fungus, Aureobasidium melanogenum, from a root system of Carya illinoinensis, and to carry out study on its plant growth-promoting effects, which provides a solid foundation for subsequent indepth development of microbial fertilizers and the reduction of chemical fertilizer usage. Another technical problem to be solved by the present invention is to provide a microbial formulation capable of promoting plant growth, and a preparation method thereof. A final technical problem to be solved by the present invention is to provide application of the endophytic fungus CNBG-PGPF-1 and a microbial formulation in promoting plant growth. Technical solutions: In order to solve the above technical problems, the present invention provides a strain of Aureobasidium melanogenum CNBG-PGPF-1. The Aureobasidium melanogenum CNBG-PGPF-1 strain was deposited with China General Microbiological Culture Collection Center (CGMCC) on May 22, 2023 under an accession number CGMCC No. 40635 in Beijing, P.R.China. The present invention further provides a microbial formulation that contains the above-mentioned Aureobasidium melanogenum CNBG-PGPF-1 or a fermentation broth filtrate thereof. The formulation includes a single formulation or a compound formulation, and a type of the formulation includes a liquid formulation or a solid formulation. A preparation method of the fermentation broth filtrate includes the following steps: obtaining mycelium plugs from freshly cultured Aureobasidium melanogenum CNBG-PGPF-1 using a puncher, placing the mycelium plugs into a potato dextrose broth (PDB) medium, collecting a fermentation broth after culturing in the dark for 710 days, filtering the fermentation broth through gauze, and diluting with sterile distilled water to obtain the fermentation broth filtrate. A composition of the PDB medium includes: 3 g potato infusion powder and 20 g glucose are added in per 1000 mL deionised water, and sterilized by high-pressure steam. The present invention also includes application of Aureobasidium melanogenum CNBG-PGPF-1 and the microbial formulation in promoting growth of a plant. In the microbial formulation, a spore concentration of Aureobasidium melanogenum CNBG-PGPF-1 is 5* 104 spores / mL - 2* 105 spores / mL. The plant includes but is not limited to one or both of Arabidopsis thaliana and tomato, and the formulation is also applicable for promoting the growth of other plants. The application specifically includes co-culture of the plant and Aureobasidium melanogenum CNBG-PGPF-1 on a solid culture medium for plant and endophytic fungi. The solid culture medium includes, but is not limited to, a Murashige and Skoog medium (MS) medium; and preferably, the solid medium is a 1 / 2 MS solid culture medium. Beneficial effects: Compared with the prior art, the present invention has the following advantages: the strain provided by the present invention can effectively promote the growth of Arabidopsis thaliana and tomato; after co-culture with the strain, the fresh weights of Arabidopsis thaliana and tomato seedlings increased by 259.18% and 182.55%, respectively, compared with the control group; the leaf area of Arabidopsis thaliana seedlings increased by 154.08% compared with the control group. After applying the fermentation broth filtrate of the strain, potted growthpromoting experiment showed that the fresh weight of the aboveground parts of tomato seedlings increased by 69.15%, the dry weight of the aboveground parts increased by 75.95%, the fresh weight of the underground parts increased by 80.00%, the dry weight of the underground parts increased by 120.01%, and the root length increased by 66.98%, compared with the control group. The application of the strain and fermentation broth filtrate thereof provided by the present invention exhibit outstanding plant growth-promoting effects, and has broad development and application prospects in the field of agricultural microbial fertilizers. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows colony morphology of a CNBG-PGPF-1 strain on a PDA medium, with a front view on a left and a back view on a right. FIG. 2 shows mycelia morphology of a CNBG-PGPF-1 strain on a PDA medium. FIG. 3 shows spore morphology of a CNBG-PGPF-1 strain on a PDA medium. FIG. 4 shows a phylogenetic tree of a CNBG-PGPF-1 strain. FIG. 5 shows a plant growth-promoting effect diagram of a CNBG-PGPF-1 strain cocultured with Arabidopsis thaliana on a 1 / 2MS medium, where a top row shows front view, and a bottom row shows a back view (Control represents a control group; and CNBG-PGPF-1 represents a treatment group). FIG. 6 shows plant growth-promoting effect data of a CNBG-PGPF-1 strain cocultured with Arabidopsis thaliana on 1 / 2MS medium, where the figure on a left side shows fresh weight data, and the figure on a right side shows leaf area data (Control represents a control group; and CNBG-PGPF-1 represents a treatment group). FIG. 7 shows a growth-promoting effect diagram of a CNBG-PGPF-1 strain cocultured with tomato (Control represents a control group; and CNBG-PGPF-1 represents a treatment group). FIG. 8 shows plant growth-promoting effect data of a CNBG-PGPF-1 strain cocultured with tomato, where the figure on a left side shows fresh weight data, and the figure on a right side shows hypocotyl length data (Control represents a control group; and CNBG-PGPF-1 represents a treatment group). FIG. 9 shows colonization of a CNBG-PGPF-1 strain on a tomato root system (Control represents a control group; and CNBG-PGPF-1 represents a treatment group). FIG. 10 shows effects of potted growth-promoting experiments using a CNBG-PGPF-1 strain on biomass of tomato seedlings, where the figure on an upper left side shows aboveground fresh weight, the figure on a top right side shows aboveground dry weight, the figure on a bottom left side shows underground fresh weight, and the figure on a bottom right side shows underground dry weight (Control represents a control group, and CNBG-PGPF-1 10%, CNBG-PGPF-1 20%, and CNBG-PGPF-1 50% are treatment groups). FIG. 11 shows effects of potted growth-promoting experiments using a CNBG-PGPF-1 strain on growth indicators of tomato seedlings, where the figure on an upper left side shows plant height data of aboveground parts, the figure on a top right side shows root length data of underground parts, the figure on a bottom left side shows SPAD data, and the figure on a bottom right side shows a number of leaves (Control represents a control group, and CNBG-PGPF-1 10%, CNBG-PGPF-1 20%, and CNBG-PGPF-1 50% are treatment groups). DETAILED DESCRIPTION OF EMBODIMENTS The present invention is further described in detail below with reference to the accompanying drawings, embodiments, and experiments. However, 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 the relevant technical field. Those of ordinary skill in the art may implement the present invention by referring to various commonly used reference books, scientific and technological literature, or relevant instructions and manuals available before the filing date of the present invention. Example 1 Isolation, purification, and morphological observation of endophytic fungus CNBG-PGPF-1 Root samples were collected from healthy pecan (Carya illinoinensis) plants grown in a planting base in Jurong City, Jiangsu Province, China, surfaces of the root samples were rinsed with tap water, and then cut into uniform segments and placed on filter paper for later use. Firstly, the cleaned samples were placed in a 10 mL centrifuge tube and repeatedly flushed and vortex-washed with sterile water. Secondly, the samples were sterilised with a 75% ethanol solution by repeated pipetting and vortex washing, and a supernatant was removed after natural sedimentation, then, the samples were rinsed three times with sterile water to eliminate the ethanol. Afterward, 10% sodium hypochlorite solution was added for disinfection, followed by repeated pipetting and vortex washing, and a supernatant was removed again after natural sedimentation. Finally, sterile water was repeatedly added to remove sodium hypochlorite by repeated pipetting and vortex washing, which was repeated three times. After sterilization, the samples were transferred in an ultra-clean bench, dissected using a sterile scalpel, and placed on a fresh potato dextrose agar (PDA) plate, and then cultured in the dark at a constant temperature of 25°C. Once mycelium grew out, fresh mycelium plugs having single colonies were picked using sterile inoculation needles based on color, texture, and growth rate of the colonies, and inoculated onto a new PDA plate, and the plate was cultured in the dark in a 25°C constant temperature incubator in an inverted position for 3-5 d. After several rounds of subcultures, a pure strain designated CNBG-PGPF-1 was obtained. The purified colonies were inoculated onto slant cultures and stored at 4°C. To observe the colonies, mycelium, and spore morphology of the CNBG-PGPF-1 strain, a PDA medium was selected for culture. PDA medium formulation: 3 g potato infusion powder, 20 g glucose, and 15 g agar were added and dissolved in 1000 mL deionized water, sterilised at 121 °C under high pressure for 20 min. Experimental results: After 5 days of culture on PDA, CNBG-PGPF-1 exhibited grayish green and flocculent mycelia with a dark green margin (FIG. 1). The mycelia showed extensive branching and were nearly transparent (FIG. 2). Spores were transparent with smooth margins, and spindle-shaped or ovoid (FIG. 3), with high sporulation density exceeding 10 spores / pL. Example 2: Molecular identification of Endophytic Fungus CNBG-PGPF-1 Firstly, plate-cultured fungi (three mycelium plugs were sufficient) were obtained using a 5 mm puncher, and placed into a 2 mL tube containing 500 pL of 0.01 M phosphate buffer (pH 7.2-7.4), and the mixture was vigorously vortex-washed. Secondly, An appropriate amount of sterilised glass beads (dia. Glass beads, BioSpec, mixed in equal proportions) with diameters of 0.5 mm and 0.1 mm were added to the tube, and the mixture was then ground with a shaker at a speed of 6.5 m / s for 1 min. Finally, the mixture was centrifuged at 12,000 rpm for 2 min, and a supernatant was taken and transferred into 1.5 mL sterilised tubes for later use. A crude DNA extract solution was used as a template for PCR amplification of an internal transcribed spacer (ITS) region using primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). A PCR reaction system was 50 pL in total volume: 25 pL EasyTaq Mix, 2 pL forward primer ITS1, 2 pL reverse primer ITS4, 3 pL DNA template, and 18 pL sterile water. PCR conditions were as follows: initial denaturation at 94°C for 5 min; 36 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 40 s; and final extension at 72 °C for 10 min. The amplified PCR products were detected by 1% agarose gel electrophoresis and then sent to Sangon Biotech for sequencing. All obtained sequences were analyzed using a Basic Local Alignment Search Tool (BLAST) search in the GenBank database of the National Center of Biotechnology Information (NCBI), and 10-15 species with highest similarity were selected for phylogenetic tree analysis. Experimental results: The sequencing results were spliced to obtain a nearly fulllength sequence of the ITS region of CNBG-PGPF-1 strain (as shown in SEQ ID NO. 1), with a total length of 550 bp. The ITS sequence was compared with homologous sequences in the GenBank database (NCBI) using BLAST, and strains with the highest similarity were identified using the BLAST. The results showed that the strain had a similarity of 98.14% to Aureobasidium melanogenum CBS. 105.22. In addition, a phylogenetic tree was constructed using the MEGA 7 software based on the Maximum Likelihood method, indicating that the strain clustered with several fungi of the genus Aureobasidium, and was closest to Aureobasidium pullulans CBS 584.75 (FIG. 4). Therefore, it can be determined that the strain was most likely a variant of Aureobasidium pullulans, namely Aureobasidium melanogenum. Afterwards, the CNBG-PGPF-1 strain was deposited with China General Microbiological Culture Collection Center (CGMCC) on May 22, 2023 under an accession number CGMCC No. 40635 at the deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, P.R.China. The strain was taxonomically designated as Aureobasidium melanogenum. Example 3 Co-culture of CNBG-PGPF-1 with Arabidopsis thaliana on 1 / 2 MS medium (1) Sterilisation of Arabidopsis thaliana seeds: a total of 60-80 Arabidopsis thaliana seeds were placed into a 2 mL centrifuge tube for surface sterilisation. Firstly, 1 mL of sterile water was used to repeatedly pipette and vortex wash the seeds for washing, and a supernatant was removed after natural sedimentation. Secondly, 1 mL of 75% ethanol was added to pipette and vortex wash the seeds for sterilisation for 2-3 min, and a supernatant was removed after natural sedimentation; and 1 mL of sterile water was added to repeatedly pipette and vortex washing to wash away residual ethanol, a supernatant was removed after natural sedimentation, which was repeated twice. Afterward, 1mL of 10% sodium hypochlorite solution was added for disinfection, followed by repeated pipetting and vortex washing for 6-8 min, and a supernatant was removed again after natural sedimentation; 1 mL of sterile water was added to repeatedly pipette and vortex washing to wash away residual sodium hypochlorite, and a supernatant was removed after natural sedimentation, which was repeated twice, and finally, 1 mL of sterile water was drawn to resuspend the seeds. (2) The sterilised Arabidopsis thaliana seeds were spread evenly on 1 / 2MS solid medium, and excess sterile water was sucked out of the medium, and then dried at the ultra-clean bench and sealed with sealing film, transferred to an illuminated incubator, and cultured vertically at 22°C for 16 h light / 8 h dark until seed germination. (3) Sterile PDA plugs (left side) and CNBG-PGPF-1 mycelium plugs (right side), each with a diameter of 5 mm and a thickness of 3 mm, were placed on the solid 1 / 2 MS medium at 0.9 cm from each edge of a Petri dish, and volumes of both PDA plugs and mycelium plugs were about 60 mm3. After germination, seedlings of uniform growth status were selected and horizontally placed on the Petri dish evenly on the Petri dish, three seedlings on each side were sealed with a sealing membrane and transferred to the illuminated incubator, and cultured vertically at 22°C for 16 h light / 8 h dark for 7-10 days. When significant differences became apparent, the Arabidopsis thaliana seedlings were collected and photographed. Experimental results: After 10 days, compared with the Arabidopsis thaliana seedlings in the control group, the Arabidopsis thaliana seedlings inoculated with CNBG-PGPF-1 exhibited larger size, greener leaves, and more developed root systems. In particular, a number of lateral roots were significantly higher than that in the control group (FIG. 5). As shown in FIG. 6, fresh weight and leaf area of the Arabidopsis thaliana seedlings inoculated with CNBG-PGPF-1 were significantly greater than those of the control group, increased by 259.18% and 154.08% respectively compared with the control group, proving obvious growth-promoting effects. Example 4 Co-culture of CNBG-PGPF-1 with tomato on 1 / 2 MS medium (1) Sterilisation of tomato seeds: a total of 60-80 tomato seeds were placed into a 15 mL centrifuge tube for surface sterilisation. Firstly, 10 mL of sterile water was used to repeatedly pipette and vortex wash the seeds for washing, and a supernatant was removed after natural sedimentation. Secondly, 10 mL of 75% ethanol was added to pipette and vortex wash the seeds for sterilisation for 2-3 min, and a supernatant was removed after natural sedimentation; and 10 mL of sterile water was added to repeatedly pipette and vortex washing to wash away residual ethanol, a supernatant was removed after natural sedimentation, which was repeated twice. Afterward, 10 mL of 10% sodium hypochlorite solution was added for disinfection, followed by repeated pipetting and vortex washing for 6-8 min, and a supernatant was removed again after natural sedimentation; 10 mL of sterile water was added to repeatedly pipette and vortex washing to wash away residual sodium hypochlorite, and a supernatant was removed after natural sedimentation, which was repeated twice, and finally, 10 mL of sterile water was drawn to resuspend the seeds. (2) The sterilised tomato seeds were spread evenly on 1 / 2MS solid medium, and excess sterile water was sucked out of the medium. They were dried at the ultra-clean bench and sealed with sealing film, labeled with a marker, and transferred to an illuminated incubator, and cultured vertically at 25°C for 12 h light / 12 h dark until radicle emergence of the tomato seeds. (3) Sterile PDA plugs (left side) and CNBG-PGPF-1 mycelium plugs (right side), each with a diameter of 5 mm and a thickness of 3 mm, were placed on the solid 1 / 2 MS medium at 0.9 cm from each edge of a Petri dish, and volumes of both PDA plugs and mycelium plugs were about 60 mm3. After radicle emergence of the tomato seeds, tomato seedlings of uniform growth status were selected and horizontally placed on the Petri dish evenly on the Petri dish, three seedlings on each side were sealed with a sealing membrane and transferred to the illuminated incubator, and cultured vertically at 25°C for 12 h light / 12 h dark for 7-12 days. When significant differences became apparent, the tomato seedlings were collected, measured, and photographed, and statistical analysis of fresh weight and hypocotyl length. Experimental results: As shown in FIGs. 7 and 8, after 12 days, tomato seedlings inoculated with the CNBG-PGPF-1 strain developed more robust root systems, and fresh weight and hypocotyl length of the tomato seedlings were significantly greater than those of the control group, increasing by 182.55% and 29.30%, respectively compared with the control group, proving obvious growth-promoting effects. During the co-culture experiment, observation under an inverted microscope revealed that CNBG-PGPF-1 hyphae could invade and extend into root tissues of the tomato seedlings. New hyphae protruded from surfaces of tomato roots in a raised pattern (FIG. 9), indicating that CNBG-PGPF-1 could successfully establish an interactive relationship with tomatoes. Example 5 Potted growth-promoting experiment of CNBG-PGPF-1 strain (1) Tomato (cultivar: Da Hong tomato) seedlings were pre-germinated. After one week, seedlings of uniform size were selected and transplanted into flowerpots (10 cm x 10 cm) filled with nutrient soil, with one seedling per flowerpot; and five flowerpots were placed in each tray as five replicates. Two trays with 10 replicates were prepared for each treatment group. (2) Freshly cultured endophytic fungi (3-5 mycelium plugs) were obtained using a 5 mm puncher, a volume of each mycelium plug was about 60 mm3, and the endophytic fungi were inoculated into 200 mL of potato dextrose broth (PDB) medium, cultured in the dark at 25°C and 160 rpm for 7 days, and a fermentation broth was collected. The fermentation broth was filtered through gauze, and then diluted with an equal volume of sterile distilled water to obtain 400 mL of fermentation filtrate with a spore concentration of lx 105 spores / mL. Four treatment groups were established for the tomatoes, that is, sterile distilled water, 10% fermentation filtrate (with a spore concentration of lx 104 spores / mL), 20% fermentation filtrate (with a spore concentration of 2* 104 spores / mL), and 50% fermentation filtrate (spore concentration: 5* 104 spores / mL). During treatment, 40 mL of solution was poured into each pot along a primary root of each plant, a total of 200 mL solution was applied for each tray. Accordingly, volumes of fermentation filtrate needed per treatment were 0 mL, 40 mL (400 mL><10%), 80 mL (400 mL><20%), and 200 mL (400 mLx50%), respectively. PDB medium formulation: 3 g potato infusion powder, and 20 g glucose were added and dissolved in 1000 mL deionized water, sterilised at 121 °C under high pressure for 20 min. (3) One week after transplanting, roots of tomato seedlings were irrigated with stock solution, the treatment was repeated once every 10 days, and repeated three times in total. During the treatment period, plant height, soil and plant analyzer development (SPAD) and other data was measured and recorded. When visible growth differences among the treatment groups could be observed with the naked eye, plant samples were collected to measure root length, fresh weight, and number of leaves. After cleaning, the plants were subjected to enzyme inactivation at 105°C for 30 min, and dried in an oven at 80°C to a constant weight, and a dry weight was measured. Experimental results: As shown in FIG. 10, CNBG-PGPF-1 fermentation broth filtrate at different concentrations all had significant plant growth-promoting effects. Specifically, the 10% fermentation broth filtrate treatment significantly increased both fresh and dry weight of aboveground parts of tomato plants by 69.15% and 75.95%, respectively, compared with the control group. The 20% fermentation broth filtrate treatment significantly increased fresh weight of underground parts of tomato plants by 80.00% compared with the control group; and the 50% fermentation broth filtrate treatment significantly increased both fresh and dry weight of underground parts of tomato plants by 120.01% compared with the control group. The measurement results further showed that the 10%, 20%, and 50% CNBG-PGPF-1 fermentation broth filtrate treatments all significantly increased root length of the tomato plants, with increases of 63.55%, 66.98%, and 50.16%, respectively, compared with to the control group. In addition, all three concentrations of filtrate treatment with three different concentrations of fermentation broth filtrate significantly increased the SPAD values of tomato plants (FIG. 11).

Claims

1. A strain of Aureobasidium melanogenum CNBG-PGPF-1, characterized in that the Aureobasidium melanogenum CNBG-PGPF-1 was deposited with China General Microbiological Culture Collection Center (CGMCC) on May 22, 2023 under an accession number CGMCC No. 40635.

2. A microbial formulation, characterized by comprising the Aureobasidium melanogenum CNBG-PGPF-1 of claim 1 or a fermentation broth filtrate thereof.

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

4. A preparation method of the microbial formulation according to claim 2 or 3, characterized in that a preparation method of the fermentation broth filtrate comprises the following steps: obtaining mycelium plugs from freshly cultured Aureobasidium melanogenum CNBG-PGPF-1 using a puncher, placing the mycelium plugs into a potato dextrose broth (PDB) medium, collecting a fermentation broth after culturing in the dark for 7-10 days, filtering the fermentation broth through gauze, and diluting with sterile distilled water to obtain the fermentation broth filtrate.

5. The preparation method of the microbial formulation according to claim 4, characterized in that a composition of the PDB medium comprises: 3 g potato infusion powder and 20 g glucose are added in per 1000 mL deionised water, and sterilised by high-pressure steam.

6. Application of the Aureobasidium melanogenum CNBG-PGPF-1 of claim 1, and the microbial formulation of claim 2 or 3 in promoting growth of a plant.

7. The application according to claim 6, characterized in that a spore concentration of Aureobasidium melanogenum CNBG-PGPF-1 in the microbial formulation is 5* 104 spores / mL - 2* 105 spores / mL.

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

9. The application according to claim 6, characterized by comprising co-culture of the plant and the Aureobasidium melanogenum CNBG-PGPF-1 on a solid culture medium for plant and endophytic fungi.

10. The application according to claim 9, characterized in that the solid culturemedium comprises a Murashige and Skoog (MS) medium.A

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