CN114196553A

CN114196553A - Aureobasidium sp MR-57 and application thereof

Info

Publication number: CN114196553A
Application number: CN202210040724.3A
Authority: CN
Inventors: 王云贺; 韩忠明; 王娇; 王妍; 杨丽华; 孙卓; 韩梅; 杨利民
Original assignee: Jilin Agricultural University
Current assignee: Jilin Agricultural University
Priority date: 2022-01-14
Filing date: 2022-01-14
Publication date: 2022-03-18
Anticipated expiration: 2042-01-14
Also published as: CN114196553B

Abstract

An aureobasidium pullulans MR-57 strain and application thereof belong to the field of functional microorganism screening and application, the strain MR-57 is preserved in Guangdong province microorganism strain preservation center at 11 and 26 months in 2021 with the preservation number GDMCC No. 62084. The strain MR-57 has stronger inhibition and antagonism to 7 pathogenic bacteria such as pillar-destroying fungi and the like, and has stronger antagonism to alternaria liriosa, pillar-destroying fungi and mechanical fungi puncturing; the strain MR-57 sterile fermentation broth has an obvious inhibition effect on the growth of fusarium equiseti hyphae and can obviously reduce the germination rate of fusarium equiseti spores; the soil mixing method is used for verifying that the strain MR-57 has good colonization capacity in soil; potted plant biocontrol tests prove that the strain MR-57 has certain effects of preventing, treating and promoting the growth of the root rot of divaricate saposhnikovia root and can obviously reduce the disease index of the root rot of divaricate saposhnikovia root.

Description

Aureobasidium sp MR-57 and application thereof

Technical Field

The invention belongs to the technical field of functional microorganism screening and application, and particularly relates to an aureobasidium pullulans (Acrophia jodhpurensis) MR-57 and application thereof.

Background

Saposhnikovia divaricata (Turcz.) Schisck. is plant of Saposhnikovia of Umbelliferae, and is administered with dry root of plant without stem of flower; pungent, slightly sweet and warm in nature, and has the effects of relieving spasm, dispelling pathogenic wind, relieving exterior syndrome, eliminating dampness and relieving pain; mainly distributed in the three provinces of northeast, the north of river, inner Mongolia and so on. In recent years, with the increase of the cultivation area of the divaricate saposhnikovia root, the divaricate saposhnikovia root rot is more and more serious in the cultivation area, the divaricate saposhnikovia root mainly infects the stem base of the divaricate saposhnikovia root, vascular bundles are damaged after the disease occurs, leaves are wilted and plants are died, and the yield and the quality of the divaricate saposhnikovia root are seriously affected. Fusarium equiseti is an important pathogenic bacterium causing divaricate saposhnikovia root rot, is a common pathogenic bacterium in tropical and subtropical regions, and gradually becomes a pathogenic bacterium which is strong in pathogenicity and can invade various hosts due to the influence of climate change.

At present, chemical pesticides are mainly used for preventing and controlling plant diseases, and the pesticides for preventing and controlling the windproof root rot mainly comprise mancozeb, hymexazol, carbendazim, thiophanate methyl and the like. Because the biological control can reduce or inhibit the occurrence of diseases, the biological control has the characteristics of greenness, safety, high efficiency, low toxicity, persistence and the like, is one of important strategies for replacing chemical control to reduce the harm of the root rot of the divaricate saposhnikovia root, accords with the 'double reduction' strategy proposed by the country, and has become the focus of research of scholars at home and abroad at present. Currently, there are pseudomonas, trichoderma fungi, bacillus, agrobacterium and the like which are developed as biocontrol bacteria and put into commercial production, but the strain resources are very limited, so how to screen and use biocontrol bacteria which can be used as biological control is a hot spot of research in recent years.

Disclosure of Invention

The invention aims to provide an aureobasidium georgii (Acrophthora jodhpurensis) MR-57 of aureobasidium and application thereof, which provide a theoretical basis for biological control of radix sileris root rot and provide a scientific basis for deep research and development of radix sileris biocontrol bacteria resources.

The technical scheme adopted by the invention for solving the technical problem is as follows:

the strain of aureobasidium pullulans MR-57 of the invention is preserved in Guangdong province microorganism strain preservation center at 11-26 months in 2021, and the preservation numbers are as follows: GDMCC No. 62084.

The invention discloses an application of an aureobasidium pullulans (Acrophthalora jodhpurensis) MR-57 in preparation of a phytopathogen antagonist.

The invention discloses an application of an aureobasidium pullulans (Acrophthalora jodhpurensis) MR-57 in preparation of a divaricate saposhnikovia pathogen antagonist.

The application of an aureobasidium geodesis (Acrophthalora jodhpurensis) MR-57 strain in preparing a rhizoctonia solani antagonist, a phytophthora infestans antagonist, a botrytis cinerea antagonist, a fusarium oxysporum antagonist, a fusarium equiseti antagonist, an alternaria tenuissima antagonist, an alternaria liriopes antagonist, a cylindracea destructor antagonist or a mechanocladium spinulosum antagonist.

The application of an aureobasidium pullulans (Acrophialophora jodhpurensis) MR-57 in preventing and treating the root rot of divaricate saposhnikovia root is disclosed.

The application of an aureobasidium pullulans (Acrophialophora jodhpurensis) MR-57 in preventing and treating the blight of divaricate saposhnikovia root is disclosed.

The invention has the beneficial effects that:

the invention screens a new microorganism from the root soil of the divaricate saposhnikovia root, performs strain identification by combining morphology, microscopic characteristics and 18 SR DNA sequence analysis, and identifies the strain MR-57 as Acrophthala jodhpurensis, which is a new Chinese record species.

The new species of the aureobasidium pullulans MR-57 screened by the invention has strong inhibition and antagonism to 7 tested pathogenic bacteria of the corynebacterium destructor, the mechanical bacterium of the thorn spore, the liriodendron strand spore, the alternaria tenuissima, the phytophthora cactorum, the botrytis cinerea and the fusarium equiseti, the inhibition rate is 60-90%, the antagonism to the corynebacterium destructor and the mechanical bacterium of the thorn spore is strong, the inhibition rates are respectively 75.22% and 73.33%, the inhibition rates to the rhizoctonia solani and the fusarium oxysporum are respectively 59.33% and 59.66%, and the bacterial strain MR-57 has a wide antibacterial spectrum.

The sterile fermentation liquid of the new species of aureobasidium pullulans MR-57 screened by the invention has obvious inhibition effect on the growth of fusarium equiseti, and can enable fusarium equiseti hyphae to be folded, broken, condensed, darkened in color, contracted, expanded, deformed, twisted and other abnormal changes; the sterile fermentation solution can obviously reduce the germination rate of fusarium equiseti spores, and has good inhibitory activity on the germination of the fusarium equiseti spores. Therefore, the strain MR-57 and the secondary metabolite thereof have the function of broad-spectrum inhibition of common phytopathogens.

The new species of aureobasidium pullulans MR-57 screened by the invention is proved to have good colonization ability in soil by a soil mixing method.

The new species of aureobasidium pullulans MR-57 screened by the invention is subjected to a pot biological control test under indoor conditions, and the result shows that the strain MR-57 has certain control and growth promotion effects on the divaricate saposhnikovia root rot, can obviously reduce the disease index of the divaricate saposhnikovia root rot, has the control effect of 65.41 percent, is equivalent to the control effect of 800 times of pesticide mancozeb, and is obviously higher than the control effects of bacillus subtilis and trichoderma harzianum; in addition, the bacterial strain MR-57 has bacteriostatic activity, and the bacterial strain MR-57 can be seen to have the effect of promoting plant growth from the biomass of the divaricate saposhnikovia root, which indicates that the bacterial strain MR-57 has certain potential for preventing and treating the root rot of the divaricate saposhnikovia root.

Drawings

In order to explain the technical solutions of the embodiments of the present application more clearly, the drawings needed to be used in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and it is obvious for those skilled in the art that other drawings can be obtained from the drawings without creative efforts.

FIG. 1 shows the morphological characteristics of the strain MR-57 of example 1 after 30 days of culture in four media, OA, PCA, CMA and MEA, respectively.

FIG. 2 is the microscopic characteristics of the conidia of strain MR-57 of example 1. In the figure, a is the microscopic characteristic of the conidium of the strain MR-57 at 20 μm, b is the microscopic characteristic of the conidium of the strain MR-57 at 1 μm, and c is the microscopic characteristic of the conidium of the strain MR-57 at 2 μm.

FIG. 3 shows the establishment of a phylogenetic tree of the strain MR-57 according to example 1 based on ITS 18.

FIG. 4 shows the inhibition rate of the strain MR-57 in example 2 against 9 pathogenic bacteria tested.

FIG. 5 is a graph showing the effect of the strain MR-57 on 9 pathogenic bacteria tested in example 2. In the figure, 1: MR-57 confronted Rhizoctonia solani, 2: MR-57 challenge Phytophthora infestans, 3: MR-57 confronts Botrytis cinerea, 4: MR-57 on Fusarium oxysporum, 5: MR-57 confronted with Fusarium equiseti, 6: MR-57 confronted Alternaria tenuissima, 7: MR-57 of Alternaria pohuashanensis, 8: MR-57 counter-standing mechanical bacterium Sporotrichum, 9: the MR-57 confronts each other to destroy the cylindrosporium.

FIG. 6 shows the growth inhibition effect of the strain MR-57 in example 3 on Fusarium equiseti hyphae. In the figure, A is the growth of Fusarium equiseti in PDA. B, the growth of the fusarium equiseti in a drug-containing culture medium containing MR-57 fermentation liquor. a is the hypha form of the fusarium equiseti grown in the PDA. b is folding, breaking and uneven thickness of fusarium equiseti hyphae. c: the inclusion is coagulated, the color becomes dark, the constriction, the expansion, the deformity and the distortion.

FIG. 7 shows the inhibitory effect of the strain MR-57 sterile fermentation broth on the germination of Fusarium equiseti spores in example 4.

Detailed Description

The screened aureobasidium pullulans (Acrophthalora jodhpurensis) MR-57 is deposited in the Guangdong province microorganism culture collection center at 26 months 11 in 2021, is called GDMCC for short, and has the address as follows: no. 59 building 5 of the Fujiu No. 100 college of the Fuzhou city of Guangdong province (microbial research institute of the academy of sciences of Guangdong province), the preservation number is: GDMCC No. 62084.

The aureobasidium pullulans (Acrophialophora jodhpurensis) MR-57 can be applied to preparation of a phytopathogen antagonist.

The aureobasidium pullulans (Acrophialophora jodhpurensis) MR-57 can be applied to preparing a divaricate saposhnikovia pathogen antagonist.

The strain of aureobasidium geodesis (Acrophthalora jodhpurensis) MR-57 can be applied to preparation of a rhizoctonia solani antagonist, a phytophthora infestans antagonist, a botrytis cinerea antagonist, a fusarium oxysporum antagonist, a fusarium equiseti antagonist, an alternaria tenuissima antagonist, an alternaria liriopes antagonist, a cylindracea antagonist or a mechanocladium spinulosum antagonist.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Test materials

The tested soil is 1 year wind-proof rhizosphere soil: is collected from a medicinal plant garden planting base of Jilin agriculture university.

Pathogenic bacteria to be tested: rhizoctonia solani, Phytophtora cactorum, Botrytis cinerea, Fusarium oxysporum, Fusarium equiseti, Alternaria tenuissima, Alternaria tulosa, Alternaria liriodendron liriodendra, Cylindrocarpon destructor, Mycocentrospora spinosa, and Mycocentrospora acarina, provided by the university of plant physiology and ecology laboratory of the college of agricultural Chinese medicinal materials.

The formulation of each medium is shown in table 1:

TABLE 1

Kit TaKaRa MiniBEST Universal Genomic DNA Extraction KitVer.5.0: purchased from Changchun Hailan Korea Co.

EXAMPLE 1 screening, identification and preservation of Strain MR-57

First, screening

(1) The tested soil is 1 year biocontrol wind rhizosphere soil, and is dried by air and then sieved by a 20-mesh sieve; weighing 10g of the sample in a 250mL triangular flask containing 90mL of sterile normal saline and glass beads; shaking for 20min, collecting 1mL supernatant, adding 9mL sterile physiological saline to obtain 10 times of dilution, and diluting to 10 times^-3、10^-4、10^-5Standby; respectively sucking 200 mul of diluent of different gradient solutions and coating the diluent on a flat plate containing a PDA culture medium, wherein each treatment is repeated for 3 times; and (3) carrying out inverted dark culture at 25 ℃ for 7 days, selecting single colonies with phenotype difference, carrying out passage 3 times, streaking on a PDA culture medium for purification culture, separating out pure colonies, and preserving at 4 ℃.

(2) Screening the strains obtained in the last step by adopting a plate confronting culture method, and screening out the fungi with bacteriostatic activity. Firstly, a puncher is used for taking fusarium equiseti cakes with the diameter of 8mm and inoculating the fusarium equiseti cakes to the center of a flat plate with the diameter of 90mm and containing a PDA culture medium, meanwhile, 2 bacterial cakes with the diameter of 8mm obtained in the last step are adhered to 2 symmetrical points 25mm away from the center of the flat plate, single inoculation pathogenic bacteria to be tested are used as a reference, the culture is carried out for 7 days at the temperature of 25 ℃, each treatment is repeated for 3 times, and the bacteriostasis rate is tested. The formula for calculating the bacteriostasis rate is as follows: (R)_C-R_P)/R_C*100％，R_CTo compare the trend radii, R_PIs the process trend radius.

6 strains are screened out by analysis, and are respectively named as MR-34, MR-37, MR-39, MR-57, MR-68 and MR-96, the bacteriostasis rate reaches over 55 percent, and the bacteriostasis rate of each strain is shown in table 2; wherein, the MR-57 has better bacteriostatic effect, and the bacteriostatic rate is about 60 percent.

TABLE 2

Bacterial strains	Rate of inhibition of bacteria
		MR-34	56.66±1.11b
MR-37	57.77±2.22ab
		MR-39	57.77±2.93ab
MR-57	60±1.92a
		MR-68	55.18±0.64b
MR-96	56.29±1.28b

II, identification

The identification method comprises culture characteristic identification, morphological characteristic identification and molecular identification, and specifically comprises the following steps:

(1) culture characterization of MR-57: OA, PCA, CMA and MEA4 culture media are selected for culture at 25 ℃, the diameter of a colony is measured after 7 days, the texture, the richness, the texture and the color of hyphae, the existence, the color and the exudate of soluble pigments and the like of the colony of MR-57 are described, and the color of the colony is determined by comparing with an International Society Color Committee (ISCC) and a national standard bureau (NBS) color name chart.

The results of the culture characterization of MR-57 are shown in FIG. 1: culturing the strain MR-57 in OA culture medium with diameter of 26-27mm, flat and sparse hypha accompanied with aerial hypha, gray front mouse of colony and black back gray; the strain MR-57 is cultured in a PCA culture medium, the diameter is 23-26mm, the hypha is velvet, the front side of the colony is white, and the back side is brown black; culturing the strain MR-57 in CMA culture medium with diameter of 22-27mm, wherein the middle part of the colony is in a velvet shape, the edge is irregular, the colony has 2-3mm white edge, the front surface of the colony is in mouse gray, and the back surface of the colony is in black; the strain MR-57 is cultured in an MEA culture medium, the diameter is 24-36mm, bacterial colonies are sequentially white, mouse gray and black from outside to inside, the bacterial colonies are gradually stacked, the back is black, and the edges are irregular.

(2) Morphological characterization of MR-57: and (3) observing the microscopic morphological characteristics of MR-57 hypha, conidium, sclerotium and the like by using a ZEISS sigma300 field emission scanning electron microscope.

The morphological feature identification and identification result of MR-57 is shown in FIG. 2: the mycelium is black brown, is partially buried and separated, and has a width of 1.5-3 μm; the conidiophores are densely covered with warts, the conidiophores are dark brown, spindle-shaped and rough in surface, and the diameter is 4.5-7 mu m multiplied by 11-14 mu m.

(3) Molecular characterization of MR-57: after culturing MR-57 in PDA culture medium for several days, extracting fungal hyphal DNA with Kit TaKaRa MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0, identifying species and genus level of fungi with ITS sequence, primers are ITS1, ITS4(ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), PCR amplification reaction system is Master Mix 12.5 mu L, ITS11 mu L, ITS 41 mu L, rDNA 2 mu L, ddH₂O8.5 μ L, amplification program 94 ℃ for 3 min; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s, and 72 ℃ for 1 min; 5min at 72 ℃; the amplified PCR product is handed to Shanghai worker for sequencing; the obtained gene sequence is submitted to GenBank, and a phylogenetic tree is established by adopting MEGA-5 software neighbor or-Joining method.

Molecular characterization of MR-57: sending the PCR amplification product of the MR-57ITS rDNA gene of the strain to a biological engineering (Shanghai) corporation for ITS sequence sequencing to obtain a sequence (SEQ ID NO.1) with the size of 482 bp; the search at NCBI revealed that the similarity was 99.38% compared with the existing sequence MN889997.1, and a phylogenetic tree was established by using MEGA-5 software Neighbor-Joining method, as shown in FIG. 3. The strain MR-57 and MN889997.1Acrophialophora jodhurensis gather into the same branch, and are identified as Acrophialophora jodhurensis by combining morphology and microscopic identification with 18S rDNA, and the obtained accession number is OK287150.1, which is a new Chinese record.

III, preservation

The screened aureobasidium pullulans (Acrophthalara jodhpurensis) MR-57 is preserved in the Guangdong province microorganism culture collection center at 11/26 th 2021, is called GDMCC for short, and has the address as follows: no. 59 building 5 of the Fujiu No. 100 college of the Fuzhou city of Guangdong province (microbial research institute of the academy of sciences of Guangdong province), the preservation number is: GDMCC No. 62084.

Example 2 broad-spectrum bacteriostatic test of Strain MR-57

9 test pathogenic bacteria of Rhizoctonia solani, Phytophthora cactorum, Botrytis cinerea, Fusarium oxysporum, Fusarium equiseti, Alternaria tenuissima, Alternaria tulosa, Alternaria liriodendra, Cylindrocarpon destructor, and Mycocentrospora mechanocarpa are used as target bacteria, and the strain MR-57 is subjected to an antibacterial spectrum test by a plate-to-plate method. The method comprises the following specific steps:

a cake of pathogenic bacteria (Rhizoctonia solani, Phytophthora parasitica calcinum, Botrytis cinerea, Fusarium oxysporum, Fusarium equiseti, Alternaria tenuissima, Alternaria liriosa, Alternaria liriodendra, Cylindrocarpon destructor, Myocoptera aculeatus) having a diameter of 8mm was taken by a punch and inoculated to the center of a plate having a diameter of 90mm containing a PDA medium, simultaneously, 2 bacterial strain MR-57 cakes with the diameter of 8mm are stuck on 2 symmetrical points at the position 30mm away from the center of the flat plate, and (3) taking single inoculated pathogenic bacteria to be tested as a control, culturing for 7d at 25 ℃, repeating each treatment for 3 times, and selecting hyphae at an antagonistic junction as shown in figure 5, microscopically observing the morphological characteristics of hyphae at the edge of a colony after the hyphae interact with the pathogenic bacteria to be tested, and detecting the inhibition effect of the strain MR-57 on different pathogenic bacteria to be tested.

The result shows that the strain MR-57 can obviously inhibit 7 tested pathogenic bacteria of pillar fungus, mechanical fungus acanthosporium, liriodendron alternata, alternaria tenuissima, phytophthora infestans, botrytis cinerea and fusarium equiseti, the bacteriostasis rate is 60-90 percent, and the specific formula is as follows: the antagonistic ability to the corynespora destructor and the mechanical bacterium spinosa is strong, the inhibition rates are respectively 75.22% and 73.33%, the inhibition rates to the rhizoctonia solani and the fusarium oxysporum are lower, and the inhibition rates are respectively 59.33% and 59.66%, so that the antibacterial spectrum of the strain MR-57 is wide (Table 2).

TABLE 2 bacteriostatic ratio of the strain MR-57 against 9 tested pathogenic bacteria

As shown in figure 4, by analyzing the average inhibition rate of the strain MR-57 to 9 pathogenic bacteria to be tested, the average inhibition effect of the strain MR-57 is found to be strong, the average inhibition rate reaches 67.2%, the mean value dispersion is moderate, and the distribution density is relatively uniform, so that the biological control mechanism of the strain MR-57 is further researched.

Example 3 experiment of strain MR-57 fermentation broth for inhibiting the growth of Fusarium equiseti hyphae

(1) Test pathogen activation and preparation of strain MR-57 fermentation broth

After the fusarium equiseti is put at room temperature and recovered for 30min, fusarium equiseti mycelium is selected under aseptic condition and respectively inoculated in the center of a flat plate containing a PDA culture medium, inverted culture is carried out at 25 ℃, and mycelium is taken for subculture until the flat plate is full of strains for the 4 th generation for later use.

Selecting single colony of the strain MR-57, inoculating the single colony into a 250mL triangular flask containing 100mL PDB culture solution, carrying out shake culture at 25 ℃ and 170r/min for 7d, filtering the obtained solution through 3 layers of gauze, and filtering the obtained filtrate through a 0.22 mu m filter membrane to obtain the strain MR-57 sterile fermentation liquor.

(2) Mixing the bacterial strain MR-57 sterile fermentation liquor and a PDA culture medium according to a volume ratio of 1:4 to prepare a drug-containing culture medium, inoculating fusarium equiseti cakes (d is 8mm) in the center of the drug-containing culture medium, carrying out opposite culture at 25 ℃ for 3d by taking a normal PDA culture medium as a reference, calculating the bacteriostasis rate, picking up pathogenic bacteria hyphae at an antagonistic junction when two bacterial colonies are mutually connected, observing the hypha morphological characteristics at the edge of a bacterial colony after interaction with the pathogenic bacteria under an optical microscope, and recording and taking pictures.

(3) The formula for calculating the bacteriostasis rate is as follows: (A)_c-A_f)/A_cX 100, Ac is the diameter of the colony grown by the pathogenic bacteria to be tested in the PDA culture medium, A_fThe bacterial colony diameter for the pathogenic bacteria to be tested is grown in a drug-containing culture medium containing a sterile fermentation liquid of the strain MR-57.

(4) The results show that: as shown in fig. 6, the bacterial strain MR-57 sterile fermentation liquor has an inhibition rate of 46.32% on fusarium equiseti, and the mycelium is picked up and microscopically observed to have folding, breaking and uneven thickness of fusarium equiseti mycelium (a), condensed content, darkened color, contracted constriction, expansion, malformation and distortion (b); the control hyphae grow uniformly, smoothly and straightly, so that the strain MR-57 generates secondary metabolites to inhibit the growth of fusarium equiseti.

Example 4 Effect of Strain MR57 fermentation broth on Fusarium equiseti spore germination test

(1) Test pathogen activation and preparation of strain MR-57 fermentation broth

After the fusarium equiseti is put at room temperature and recovered for 30min, fusarium equiseti mycelium is selected under aseptic condition and respectively inoculated in the center of a flat plate containing a PDA culture medium, inverted culture is carried out at 28 ℃, and mycelium is taken for subculture until the flat plate is full of strains for the 4 th generation for later use.

Selecting single colony of the strain MR-57, inoculating the single colony into a 250mL triangular flask containing 100mLPDB culture solution, performing shake culture at 25 ℃ and 170r/min for 7d, filtering the single colony through 3 layers of gauze, and filtering the obtained filtrate through a 0.22 mu m filter membrane to obtain the sterile fermentation liquor of the strain MR-57.

(2) Preparing fusarium equiseti conidia into 1 × 10 by using sterile water⁶CFU/ml, then mixing fusarium equiseti spore suspension and the bacterial strain MR-57 sterile fermentation broth uniformly in a volume ratio of 1:1, mixing fusarium equiseti spore suspension and a PDB culture medium uniformly in a volume ratio of 1:1 as a control, culturing at 25 ℃, treating for 6h, 12h, 24h and 48h, and then performing microscopic examination on fusarium equiseti spore germination on a glass slide.

(3) The germination condition of the fusarium equiseti spores treated by the strain MR-57 sterile fermentation liquid is observed by a microscope, and the result shows that the highest inhibition rate of the strain MR-57 on the germination of the fusarium equiseti spores at 6h is 90.59%, the inhibition rate of the spore germination after 24h is 60.95%, and the inhibition rate of the spore germination after 48h is 71.02%, as shown in figure 7.

EXAMPLE 5 Strain MR-57 screening for rifampicin resistance and in-soil colonization assay

(1) Subjecting the strain MR-57 to rifampicin resistance mutagenesis, sequentially culturing the strain MR-57 in a rifampicin PDB medium containing rifampicin at concentrations of 50, 100, 200, 300 μ g/ml, and gradually subjecting to rifampicin resistance mutagenesis to obtain the strain MR_Rif-57; comparison of the Strain MR-57 with the Strain MR_Rif-57 bacteriostatic activity against fusarium oxysporum and fusarium equiseti; inoculating the soil with the concentration of 1 × 10⁷Strain MR of CFU/ml_Rif30ml of 57 spore suspension, uniformly mixing with soil, placing in a room, taking soil samples every 7 days, and taking 10g of soil samples for 3 times; taking a soil sample for 5 times, adding 10g of the soil sample into 90ml of sterile raw distilled water, uniformly mixing and standing; diluting 3 times by gradient dilution method, spreading 200 μ L of the final 1 dilution on PDA plate containing 300 μ g/ml rifampicin, placing in 25 deg.C incubator for 3d, counting and recording colony number.

(2) Strain MR_RifAfter 10 generations of inoculation, 57 can stably grow on PDA plates containing 300 muL/mg of rifampicin, and has no obvious change compared with the shape of the strain MR-57,the strain MR-57 is proved to have genetic stability; strain MR_RifAfter 57 is confronted with fusarium equiseti for 7 days, the bacteriostasis rate is 60 percent; the antibacterial rate of the strain is equal to that of the strain MR-57 and the fusarium equiseti confront for 7d, which shows that the rifampicin mark has no significant influence on the antibacterial capacity of the strain MR-57; after the strain MR-57 is colonized in soil, the bacteria content of the soil tends to decrease first and then increase and finally decrease slowly, and the maximum bacteria content is 2.83 multiplied by 10 on day 21⁵The bacteria content of the soil can still reach 1.92 multiplied by 10 after 35 days of CFU/ml⁵CFU/ml shows that the strain MR-57 has good colonization effect in soil, and can be used for potted plant disease prevention experiments, and the results are shown in Table 3.

TABLE 3 colonisation of soil by MR-57 (10)⁵CFU/g)

Time/d	0	7	14	21	28	35
							Strain MR-57	10	1.8	1.97	2.83	2.22	1.92

Example 6 outdoor prevention of root rot of Saposhnikovia divaricata and its growth promoting effects of the Strain MR-57

(1) Taking 1 year-old biocontrol wind plants with consistent growth vigor to perform a potting experiment, and processing 7 pots each time; preparing spore suspension including Fusarium equiseti, Bacillus subtilis, Trichoderma harzianum, and strain MR-57 at concentration of 1 × 10⁷CFU/ml, the pesticide mancozeb is 800 times of liquid; the pot experiment was set up for five treatments: the group A is a group of single inoculated fusarium equiseti, the group B is inoculated with fusarium equiseti and trichoderma harzianum, the group C is inoculated with fusarium equiseti and bacillus subtilis, the group D is inoculated with fusarium equiseti and sprayed with pesticide mancozeb, and the group E is inoculated with fusarium equiseti and the strain MR-57; taking 1-year-old divaricate saposhnikovia roots with consistent growth vigor, scratching the rhizome parts by adopting a root-damaging irrigation method, inoculating 10ml of fusarium equiseti spore suspension, and then respectively inoculating 10ml of MR-57 spore suspension, 50ml of mancozeb, 50ml of bacillus subtilis and 50ml of trichoderma harzianum; and performing conventional management, counting the disease degree and biomass indexes of the height, the length, the fresh weight, the whole fresh weight, the dry weight and the dry weight of the roots of the divaricate saposhnikovia root after 30 days, and calculating the disease index and the control effect.

(2) The incidence of the root rot of divaricate saposhnikovia root is classified into grade 9; level 0: healthy plants without disease spots; level 1: leaf development of less than 10% of the whole plant; and 3, level: the disease occurs in 11-25% of leaves of the whole plant; and 5, stage: 26-50% of leaves of the whole plant are attacked; and 7, stage: 51-75% of leaves of the whole plant are attacked; and 9, stage: more than 76% of leaves of the whole plant develop.

(3) The disease index [ Σ (number of disease-grade plants × representative value)/(total number of plants × highest disease-grade representative value) ] × 100;

the preventing and treating effect (%) is (contrast disease index-treatment disease index)/contrast disease index x 100.

(4) The bacterial strain MR-57 has the following effects of preventing and treating the root rot of the divaricate saposhnikovia root: as can be seen from Table 4, the strain MR-57 has a good disease prevention effect which can reach 65.41%.

Potted plant disease prevention experiments show that the strain MR-57 has good disease prevention effect (Table 4). The disease index is 22.22, which is obviously lower than that of fusarium equiseti; the control effect of the strain MR-57 is 65.41% which is equivalent to 61.54% of the control effect of pesticide mancozeb, and is obviously better than the control effect of the microbial inoculum of bacillus subtilis 47.82% and trichoderma harzianum 51.89%.

TABLE 4 different treatment effects on control of root rot of Saposhnikovia divaricata potted plants

	Index of disease condition	Prevention effect
			Fusarium equiseti (CK)	64.14±2.18a
Trichoderma harzianum and fusarium equiseti	30.85±4.29bc	51.89±6.7b
			Bacillus subtilis and fusarium equiseti	32.07±5.66b	49.99±8.82b
Mancozeb + fusarium equiseti	24.66±2.11cd	61.54±3.3ab
			Strain MR-57+ fusarium equiseti	22.18±3.72d	65.41±5.8a

The fresh weight, the root fresh weight, the plant dry weight and the root dry weight of the divaricate saposhnikovia root treated by the strain MR-57 are obviously different (table 5), the fresh weight and the dry weight of the whole divaricate saposhnikovia root treated by the strain MR-57 respectively reach 20.18g and 4.94g, which are obviously higher than those of other treatment groups; the fresh and dry root weights reached 6.64g and 1.42g, significantly higher than the other treatment groups.

TABLE 5 growth promoting effect of different treatments on Saposhnikovia divaricata pot culture

Length of plant cm

Root length cm

Fresh weight of plant g

Fresh weight of root g

Dry weight g of plant

Dry root weight g

Fusarium equiseti (CK)

52.01±7.89a

29.88±4.75a

9.49±3.26b

2.66±0.19c

2.41±0.78b

0.56±0.07b

Mancozeb

53.54±2.21a

29.36±1.72a

10.81±2.84b

3.39±0.34bc

2.48±0.57b

0.73±0.24b

Trichoderma harzianum

53.9±0.49a

29.03±0.49a

11.97±0.23b

3.58±0.14b

3.18±0.14b

0.86±0.09b

Bacillus subtilis

56.64±1.6a

30.34±0.24a

10.44±0.79b

3.43±0.42bc

2.56±0.72b

0.71±0.07b

Strain MR-57

55.93±6.9a

30.93±4.05a

20.18±1.59a

6.64±1.13a

4.94±0.19a

1.42±0.02a

Example 7 outdoor control of Ledebouriella wilt with Strain MR-57

(1) Taking 1 year-old biocontrol wind plants with consistent growth vigor to perform a potting experiment, and processing 7 pots each time; preparing fusarium oxysporum, bacillus subtilis, trichoderma harzianum and strain MR-57 of spore suspension, wherein the concentrations are all 1 multiplied by 10⁷CFU/ml, and 500 times of pesticide carbendazim solution; the pot experiment was set up for five treatments: the group A is used for single inoculation of fusarium oxysporum, the group B is used for inoculation of fusarium oxysporum and trichoderma harzianum, the group C is used for inoculation of fusarium oxysporum and bacillus subtilis, the group D is used for inoculation of fusarium oxysporum and spraying of pesticide carbendazim, and the group E is used for inoculation of fusarium oxysporum and strain MR-57; taking 1-year-old divaricate saposhnikovia roots with consistent growth vigor, scratching root parts by adopting a root-damaging irrigation method, inoculating 10ml of fusarium oxysporum spore suspension, and then respectively inoculating 10ml of MR-57 spore suspension, 50ml of mancozeb, 50ml of bacillus subtilis and 50ml of trichoderma harzianum; and performing conventional management, counting the disease degree and biomass indexes of the height, the length, the fresh weight, the whole fresh weight, the dry weight and the dry weight of the roots of the divaricate saposhnikovia root after 30 days, and calculating the disease index and the control effect.

(2) The disease degree of the divaricate saposhnikovia root wilt is classified into 9 grades. Level 0: no disease symptoms exist; level 1: yellow spots are formed on the surface of the leaf and account for 1-10% of the area of the leaf; and 3, level: the edges of the leaves are curled up to be withered, withered disease regions on the leaves are obvious, the color of the disease regions is deepened, and the disease regions account for 11-25% of the whole leaf area; and 5, stage: the disease degree gradually deepens, leaves fall off due to severe withering, and 26% -50% of diseases occur; and 7, stage: the leaves of the plants fall off, the disease area continues to expand, and the area reaches 51-75%; and 9, stage: when the blight of the plants reaches the most serious, more than 75% of the leaf surfaces are affected by the blight, most of the leaf surfaces have blight spots, the color is brown, the phenomenon that the leaves fall integrally is increased, and some whole plants are withered and die.

(3) Disease index ═ Σ (number of diseased plants at each stage × representative value at each stage)/(total number of investigated plants × highest representative value) × 100%;

the prevention and treatment effect is (contrast disease index-treatment disease index)/contrast disease index x 100%.

(4) The bacterial strain MR-57 has the following effects of preventing and treating the blight of divaricate saposhnikovia root: as can be seen from Table 5, the strain MR-57 has a good disease prevention effect, and the prevention effect can reach 61.05%.

Potted plant disease prevention experiments show that the strain MR-57 has good disease prevention effect (Table 5). The disease index is 19.7, which is significantly lower than the control; the treatment and prevention effect of the strain MR-57 is 61.05 percent equivalent to the treatment effect of pesticides carbendazim and trichoderma harzianum.

TABLE 5 control effect of different treatments on the Fang Feng wilt disease of potted plants

	Disease index%	Control effect%
			Fusarium equiseti (CK)	50.59±4.29a	-
Carbendazim	16±4.23b	68.37±8.36a
			Bacillus subtilis	22.18±3.72b	56.15±7.35b
Trichoderma harzianum	16±4.23b	68.37±8.36ab
			MR-57	19.7±2.18b	61.05±4.31ab

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Jilin university of agriculture

<120> aureobasidium pullulans MR-57 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 482

<212> DNA

<213> Artificial (Primer sequences)

<400> 1

ctccccaacc attgtgacct taccttctac cgttgcttcg gcgggcgggc cacagcgccc 60

cccgggcccc ctcgggggcg cccgccggag gataaccaaa ctcttgatat tcatggcctc 120

tctgagtctt ctgtactgaa taagtcaaaa ctttcaacaa cggatctctt ggttctggca 180

tcgatgaaga acgcagcgaa atgcgataag taatgtgaat tgcagaattc agtgaatcat 240

cgaatctttg aacgcacatt gcgcccgcca gtattctggc gggcatgcct gttcgagcgt 300

catttcaacc atcaagcccc cggcttgtgt tggggacctg cggctgcccg caggccctga 360

aaaccagtgg cgggctcgct gtcacaccga gcgtagtagc aacatctcgc tcagggcgtg 420

ctgcgggttc cggccgttaa aagcctttta cacccaaggt gacctcggat caggtagaga 480

cc 482

Claims

1. An aureobasidium pullulans (Acrophthalara jodhpurensis) MR-57 is characterized in that the strain is preserved in the Guangdong province collection of microorganism strains at 26 months 11 and 2021, and the preservation numbers are as follows: GDMCC No. 62084.

2. Use of an aureobasidium pullulans (Acrophthalara jodhpurensis) MR-57 according to claim 1 for the preparation of a phytopathogen antagonist.

3. Use of an aureobasidium pullulans (Acrophthalara jodhpurensis) MR-57 according to claim 1 for the preparation of an antagonist against pathogenic wind-weed.

4. Use of an A. terreus (Acrophthalora jodhpurensis) MR-57 as claimed in claim 1 for the preparation of an antagonist against Rhizoctonia solani, an antagonist against Phytophthora infestans, an antagonist against Botrytis cinerea, an antagonist against Fusarium oxysporum, an antagonist against Fusarium equiseti, an antagonist against Alternaria tenuissima, an antagonist against Alternaria lividiana, an antagonist against Stylotrichum destructor or an antagonist against Acinetobacter aculeatus.

5. Use of an aureobasidium pullulans (Acrophthalara jodhpurensis) MR-57 according to claim 1 for controlling root rot of Saposhnikovia divaricata.

6. The use of an aureobasidium pullulans (Acrophthalara jodhpurensis) MR-57 as claimed in claim 1 for the control of Leptosphaeria nodorum.