Saccharomyces cerevisiae and application thereof

Abstract

The invention discloses trichosporon and application thereof, and relates to the technical field of phytopathogen prevention and control. The trichosporon sp. Is named as trichosporon sp., the strain number of the trichosporon sp. Is ZJU2023TR1, and the preservation number of the trichosporon sp. Is CCTCC (China Center for Type Culture Collection) NO: M 20231367. The trichosporon sp. ZJU2023TR1 provided by the invention can inhibit the growth of pyricularia grisea and corn southern leaf blight pathogenic bacteria in a plate confrontation test; in a fermentation liquor test, the inhibition effect on magnaporthe oryzae reaches up to 87.65%, and the inhibition effect on corn southern leaf blight pathogenic bacteria reaches up to 91.58%. Therefore, the strain ZJU2023TR1 can effectively inhibit the growth of the pyricularia grisea and the corn southern leaf blight pathogenic bacteria, can be used for preparing a novel, efficient and broad-spectrum biocontrol inoculant, and provides a new way for the development of microorganisms applied to biological control.

Description

Technical Field

[0001] The invention relates to the technical field of plant pathogen prevention and control, in particular to a trichosporon and an application thereof. Background Art

[0002] Rice blast, also known as rice fever, is one of the important diseases of rice. The rice disease varies greatly due to different varieties, cultivation management and climatic conditions. In the years when rice blast is prevalent, it can generally lead to a 10% to 20% reduction in rice production. In severe years, it can reduce production by 40% to 50% or even cause a total crop failure in some fields. Rice blast can occur throughout the entire growth period of rice. According to the location of the rice disease, it can be divided into seedling blast, leaf blast, ear neck blast, grain blast, etc. The rice blast fungus is the pathogen of rice blast. It has the characteristics of a wide range of hosts, easy mutation to produce new physiological races, strong resistance of conidia and mycelium, etc., which makes rice blast surpass sheath blight and white leaf blight to become the first of the three major diseases. The pathogen of rice blast is Magnaporthe oryzae. At present, the main measures for preventing and controlling rice blast include selecting disease-resistant rice varieties, using appropriate amounts of chemical pesticides, strengthening cultivation management, enhancing water and fertilizer management, and treating diseased rice straw. However, these measures all have certain limitations in prevention and control. Researchers are required to continuously explore and innovate in the existing rice blast prevention and control system, and continuously explore new potential genes and green pesticides, so as to ensure high and stable rice yields and maximize their social benefits.

[0003] Corn leaf blight is one of the important diseases in corn production. The disease is widely distributed and common. The occurrence in some local areas is heavier, which has a serious impact on corn production. In some warm and humid areas of my country, such as the summer corn area of ​​Huanghuaihai, corn leaf blight is common and more serious in some areas. The pathogen of corn leaf blight is Cochliobolusheterostrophus, which mainly harms leaves, but also sheaths, bracts and ears; the fungus can occur throughout the growth period of corn, but the disease is more serious during the tasseling and filling stages. Corn leaf blight is common and serious in the warm and humid areas of the Yellow River and Yangtze River basins in my country, generally causing a 15-20% reduction in yield, and a severe reduction of more than 50%, or even no harvest. Generally, the summer corn area has a more serious occurrence, and the year of the epidemic can cause significant losses in yield.

[0004] Biological control refers to the use of organisms and their metabolites to control plant diseases and insect pests. Its essence is to use the inter-species and intra-species relationships of organisms to adjust the density of harmful organisms, that is, the control of the biological community by the biological community. Compared with traditional control technologies, biological control technology has the advantages of no pollution to the environment, safety to humans and other organisms, no product residues, easy coordination with other plant protection measures and energy saving, and plays an increasingly important role in the integrated control of pests. So far, fungi, bacteria, actinomycetes and yeasts can be used for biological control.

[0005] In the actual production process, traditional pesticide control is common. However, according to literature reports, the pesticide utilization rate is generally 10%, and about 90% of pesticides remain in the environment, causing environmental pollution. In addition, pesticide residues can also cause harm to humans and animals. Therefore, screening beneficial microorganisms for biological control is an important method to improve the safety of crop production. Summary of the invention

[0006] Based on this, the present invention provides a trichosporon and its application. The strain ZJU2023TR1 of the present invention is isolated from the soil of Zijingang Campus of Zhejiang University, Hangzhou, Zhejiang Province (30.312106 degrees north latitude, 120.097711 degrees east longitude). Through sequence comparison, the ZJU2023TR1 strain is closest to the Trichosporon sp. sequence, indicating that the ZJU2023TR1 strain isolated by us is a trichosporon (Trichosporon sp.).

[0007] The invention provides a trichosporon, which is named as Trichosporon sp., with strain number ZJU2023TR1 and preservation number CCTCC NO: M 20231367.

[0008] The present invention also provides the use of the trichosporon in preparing a plant pathogen inhibitor, wherein the plant pathogen is Magnaporthe oryzae or Cochliobolus erythrostrophus.

[0009] The invention also provides a plant pathogen inhibitor, wherein the active ingredient comprises the Trichosporon or the fermentation liquid of the Trichosporon.

[0010] The ZJU2023TR1 strain was cultured against the pathogen of corn leaf blight and the rice blast pathogen, and the mycelial growth inhibition rates of the two pathogens could reach 25.62% and 24.62%, respectively; when cultured on a fermentation broth medium containing the ZJU2023TR1 strain, the inhibitory effect on the corn leaf blight pathogen and the rice blast pathogen was obvious.

[0011] Preferably, the volume percentage concentration of the fermentation broth of Trichosporon is 10% to 40%.

[0012] When the plant pathogen is the pathogen of corn leaf blight, the volume percentage concentration of the fermentation liquid of the trichosporon is 10% to 30%. When culture is carried out on a fermentation liquid culture medium with this volume percentage concentration, when 10% of the fermentation liquid is added, the inhibitory effect on the pathogen of corn leaf blight is as high as 66.71%. When 30% of the fermentation liquid is added, the inhibitory effect on the pathogen of corn leaf blight is basically as high as 91.58%.

[0013] When the plant pathogen is rice blast fungus, the volume percentage concentration of the fermentation liquid of the trichosporon is 10% to 40%. When culture is carried out on a fermentation liquid culture medium with this volume percentage concentration, when 10% of the fermentation liquid is added, the inhibitory effect on rice blast fungus is as high as 12.55%. When 40% of the fermentation liquid is added, the inhibitory effect on rice blast fungus is basically as high as 87.65%.

[0014] The present invention also provides a method for preparing the plant pathogen inhibitor. When the active ingredient of the plant pathogen inhibitor includes the fermentation liquid of the Trichosporon, the preparation method is:

[0015] After the Trichosporon is inoculated into a fermentation medium and cultured, the fermentation liquid after removing the bacterial bodies is the plant pathogen inhibitor. The culture temperature is 25° C. and the culture is carried out for 4 days.

[0016] The present invention also provides the use of the trichosporon or the plant pathogen inhibitor in inhibiting the growth of plant pathogens, wherein the plant pathogens are rice blast pathogens or corn leaf blight pathogens.

[0017] The present invention also provides a method for preventing and controlling plant pathogens, which comprises spraying the Trichosporon or the plant pathogen inhibitor on plants, wherein the plants are rice or corn.

[0018] Beneficial effects of the present invention:

[0019] The Trichosporon ZJU2023TR1 provided by the present invention can inhibit the growth of rice blast fungi and corn leaf blight pathogens in a plate confrontation test; in a fermentation liquid test, the inhibitory effect on rice blast fungi is as high as 87.65%, and the inhibitory effect on corn leaf blight pathogens is as high as 91.58%. It shows that the strain ZJU2023TR1 can effectively inhibit the growth of rice blast fungi and corn leaf blight pathogens, can be used to prepare a new, efficient, broad-spectrum biocontrol agent, and provides a new way for the development of microorganisms in biological control. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1The colony morphology of Trichosporon ZJU2023TR1 grown on PDA medium for 8 days; wherein A is the front view of the culture dish, and B is the back view of the culture dish.

[0021] Figure 2 This is the hyphae morphology of Trichosporon ZJU2023TR1.

[0022] Figure 3 The graph is a test result of the inhibition of Trichosporon ZJU2023TR1 on the pathogen of corn leaf blight; wherein, A is a graph of the inhibition of Trichosporon ZJU2023TR1 on the pathogen of corn leaf blight, and B is the growth status of the pathogen of corn leaf blight.

[0023] Figure 4 Graph showing the results of the inhibition of Trichosporon ZJU2023TR1 on rice blast fungus; wherein, A is a graph showing the inhibition of Trichosporon ZJU2023TR1 on rice blast fungus, and B is a graph showing the growth of rice blast fungus.

[0024] Figure 5 These are a test graph and a bar graph of the inhibitory effect of the fermentation liquid of Trichosporon ZJU2023TR1 on the pathogen of corn leaf blight; wherein, A is a test graph of the inhibitory effect of the fermentation liquid of Trichosporon ZJU2023TR1 on the pathogen of corn leaf blight, and B is a bar graph of the inhibitory effect of the fermentation liquid of Trichosporon ZJU2023TR1 on the pathogen of corn leaf blight.

[0025] Figure 6 These are a test graph and a bar graph of the inhibitory effect of the fermentation liquid of Trichosporon ZJU2023TR1 on rice blast fungus; wherein, A is a test graph of the inhibitory effect of the fermentation liquid of Trichosporon ZJU2023TR1 on rice blast fungus, and B is a bar graph of the inhibitory effect of the fermentation liquid of Trichosporon ZJU2023TR1 on rice blast fungus. DETAILED DESCRIPTION

[0026] Example 1: Bacterial strain separation

[0027] (1) Soil samples were collected from the mulberry garden of the Zijingang Campus of Zhejiang University in Hangzhou, Zhejiang Province, and separated using the selective medium dilution plate method. The process was as follows: Take four 1.5 mL centrifuge tubes and mark them with a marker pen. -3 , 10 -4 , 10 -5 , 10 -6 , use a pipette to draw 0.9 mL of sterile water and add it to each centrifuge tube;

[0028] (2) Weigh 1 g of sample and place it in a stoppered test tube containing 9 mL of sterile water. Oscillate for 10 min. -1 of soil dilution;

[0029] (3)10 -1 After shaking the soil dilution, let it stand for 2 minutes. Use a pipette to draw 0.1 mL of the suspension and add it to a centrifuge tube containing 0.9 mL of sterile water. Dilute it to make 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 diluent;

[0030] (4) Pour the sterilized PDA medium (streptomycin and ampicillin are added to the medium to reach 30 μg / L to inhibit bacterial growth) cooled to 50-60°C into the culture dishes, 20 mL per dish; after the culture medium cools to a flat plate, use a pipette to take 10 -6 , 10 -5 , 10 -4 , 10 -3 Add 0.1 mL of the diluent suspension to the plate, and then spread each dilution evenly with a sterile spreading stick, repeating 3 plates for each concentration;

[0031] (5) Place the culture dish upside down in a constant temperature incubator at 25°C for 3-5 days and then pick out the hyphae of a single colony and place it on a new plate.

[0032] PDA medium: 200g potato, 10g glucose, 15g agar powder, add distilled water to 1000ml, pH natural. Autoclave at 121℃ for 15min.

[0033] A strain was isolated from the soil of Zijingang Campus of Zhejiang University, Hangzhou, Zhejiang Province (30.312106 degrees north latitude, 120.097711 degrees east longitude) and named ZJU2023TR1.

[0034] Example 2: Identification of bacterial species morphology

[0035] After 8 days of growth in PDA medium at 25℃ in the dark, the diameter of the colony is between 36.32 and 42.64 mm, and the average growth rate of mycelium is 4.54 to 5.33 mm per day. After 13.13 to 15.44 days, the plate with a diameter of 70 mm can be fully grown. The colony as a whole spreads outward in an irregular shape, and sometimes several irregular small colonies of different sizes can be formed. The front of the colony is basically cheese white ( Figure 1 A), the back is pale yellowish orange ( Figure 1B). The colony morphology near the center of the front is porous, wrinkled, and gyrus-like, while the colony morphology away from the center of the front gradually becomes smooth and extends irregularly around; the colony on the back is smooth and has no special morphology. No conidia of ZJU2023TR1 were found under a 100x microscope, but its dense and abundant hyphae were observed. The hyphae of the fungus were slender and uniform, slightly pointed at the end, and no hyphae separation was found under a 100x microscope ( Figure 2 ).

[0036] Based on the comprehensive characteristics of colony morphology, hyphae morphology and the results of molecular biological identification, strain ZJU2023TR1 was identified as Trichosporon.

[0037] Example 3: Molecular identification

[0038] (1) DNA extraction

[0039] 1) After culturing ZJU2023TR1 strain on a PDA plate at 25°C for 7 days, scrape the mycelium on the plate with a toothpick and place it in a sterilized 1.5 mL centrifuge tube containing 500 μL DNA extraction buffer and an appropriate amount of quartz sand;

[0040] The formula of the extraction buffer was: 10 mM EDTA, 100 mM Tris-HCl, 1 M KCl;

[0041] 2) Place the centrifuge tube in the MP -24 homogenizer, oscillate at 65 Hz for 2 min to break up the mycelial tissue;

[0042] 3) Centrifuge at 12000 rpm for 10 min, pipette 300 μL of supernatant and inject into a new centrifuge tube;

[0043] 4) Add isopropanol of equal volume to the supernatant, mix by inverting, and place the centrifuge tube in a 4°C refrigerator for 10 minutes to precipitate the DNA better;

[0044] 5) Centrifuge at 12000 rpm for 10 min, discard the supernatant and keep the precipitate;

[0045] 6) Add 800 μL of 70% ethanol (v / v) to dissolve impurities, mix gently, and centrifuge at 12,000 rpm for 5 min;

[0046] 7) Discard the supernatant and place the centrifuge tube upside down on absorbent paper to evaporate excess ethanol. The white precipitate is the genomic DNA to be extracted. Add 50 μL of sterile water and dissolve it at room temperature for 15 minutes. After the dissolution is completed, store the genome in a -20°C refrigerator.

[0047] (2) PCR amplification of fungal ribosomal ITS rDNA gene and 28S ribosomal large subunit rRNA gene (LSU).

[0048] ITS primers: the sequence of the upstream primer ITS1 is: 5′-TCCGTAGGTGAACCTGCGG-3′, the sequence of the downstream primer ITS4 is: 5′-TCCTCCGCTTATTGATATGC-3′;

[0049] LSU primers: the sequence of the upstream primer LSU-F is: 5′-GTACCCGCTGAACTTAAGC-3′, and the sequence of the downstream primer LSU-R is: 5′-TCCTGAGGGAAACTTCG-3′;

[0050] PCR amplification was performed in a 20 μL reaction system, which contained: 1 μL of upstream and downstream primers, 10 μL of Green Taq Mix, 2 μL of template DNA, and ddH 2 2 μL of HO.

[0051] PCR amplification was performed on a GE-TOUCH intelligent gradient gene amplification instrument. Reaction conditions: 95°C pre-denaturation for 3 min, followed by 35 cycles including: 95°C denaturation for 30 s, 58°C annealing for 30 s, 72°C extension for 1 min, and finally 72°C post-extension for 10 min.

[0052] (3) Gene sequencing and sequence analysis

[0053] The target DNA fragments were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing. After strict verification of the sequencing results, the DNA fragment sequences shown in SEQ ID No. 1 and SEQ ID No. 2 were obtained, which were the PCR amplification products of the fungal ribosomal ITS rDNA gene and the 28S ribosomal large subunit rRNA gene (LSU).

[0054] On the NCBI website, the nucleotide sequence of the ITS of the ZJU2023TR1 strain and the 28S ribosomal large subunit rRNA gene (LSU) sequence were measured and compared with homologous or similar nucleotide sequences in the GenBank database using BLAST. Through sequence comparison, the ZJU2023TR1 strain was closest to the Trichosporon sp. sequence, and this result was the same as the morphological identification result, indicating that the ZJU2023TR1 strain we isolated was a Trichosporon sp. The newly screened strain was named Trichosporon sp., with the strain number ZJU2023TR1, and was deposited in the China Center for Type Culture Collection at Wuhan University, China on July 24, 2023, with the deposit number CCTCC NO: M 20231367.

[0055] Example 4: Inhibitory effect of strain ZJU2023TR1 on the pathogen of corn leaf blight

[0056] The ZJU2023TR1 strain was cultured in opposition to the corn leaf blight pathogen: On a PDA plate with a diameter of 90 mm, the corn leaf blight pathogen and the ZJU2023TR1 strain were inoculated on both sides of the plate, with a distance of 5 cm between the two strains. After the ZJU2023TR1 strain and the corn leaf blight pathogen were cultured in opposition on the PDA plate for 7 days, the growth of the leading edge of the corn leaf blight pathogen colony in the direction close to the ZJU2023TR1 strain was inhibited, and the hyphae showed a significant inhibition state ( Figure 3 ) According to the measurement and calculation, the mycelium growth inhibition rate can reach 25.62% (growth inhibition rate = (radius of corn leaf blight pathogen colony - radius of corn leaf blight pathogen colony in confrontation culture) / radius of corn leaf blight pathogen colony).

[0057] Example 5: Inhibitory effect of ZJU2023TR1 strain on rice blast fungus

[0058] ZJU2023TR1 strain and rice blast fungus were cultured in confrontation: On a PDA plate with a diameter of 90 mm, rice blast fungus and ZJU2023TR1 strain were inoculated on both sides of the plate, with the two strains 4.5 cm apart. After ZJU2023TR1 strain and rice blast fungus were cultured in confrontation on the PDA plate for 8 days, the growth of the leading edge of the rice blast fungus colony in the direction close to the ZJU2023TR1 strain was inhibited, and the hyphae showed a significant inhibition state ( Figure 4 ) According to the measurement and calculation, the mycelium growth inhibition rate can reach 24.62% (growth inhibition rate = (rice blast fungus colony radius - confrontation culture rice blast fungus colony radius) / rice blast fungus colony radius).

[0059] Example 6: Detection of the inhibitory effect of ZJU2023TR1 fermentation broth on corn leaf blight pathogen

[0060] The ZJU2023TR1 strain was inoculated in 100mL PDA medium / 250mL conical flask, cultured at 25℃, 150rpm for 4 days, and the supernatant was centrifuged as the fermentation broth. After filtration and sterilization, it was added to the PDA medium in proportion to make a fermentation broth-PDA plate, inoculated with corn leaf blight pathogens, and the antibacterial effect was observed for 4 days. The corn leaf blight pathogen was inoculated on PDA medium containing no fermentation broth, 10%, 20%, and 30% fermentation broth (v / m) and cultured for 4 days. Three replicates were set for different concentrations in each group. Figure 5 As shown in A, the higher the concentration of the fermented liquid, the more obvious the inhibitory effect on the pathogen of corn leaf blight. Figure 5 As shown in the bar graph B, when 10% of the fermentation liquid was added, the inhibition effect was as high as 66.71%, and when 30% of the fermentation liquid was added, the inhibition effect was basically as high as 91.58%.

[0061] Example 7: Detection of the inhibitory effect of ZJU2023TR1 fermentation broth on rice blast fungus

[0062] ZJU2023TR1 strain was inoculated in 150mL PDA medium / 250mL conical flask, 25℃, 150rpm, and cultured for 4 days. The supernatant was centrifuged as the fermentation broth, which was filtered and sterilized and added to the CM medium in proportion to make a fermentation broth-CM plate, which was inoculated with rice blast fungus and the antibacterial effect was observed for 4 days. The rice blast fungus was inoculated on CM medium without fermentation broth, 10%, 20%, 30%, and 40% fermentation broth (v / v) and cultured for 4 days. Three replicates were set at different concentrations in each group ( Figure 6 ).like Figure 6 As shown in A, the higher the concentration of the fermentation liquid, the more obvious the inhibitory effect on rice blast fungus, while the control group had no inhibitory effect on rice blast fungus. Figure 6 As shown in B, when 10% fermentation broth was added, the inhibition effect was as high as 12.55%. When 40% fermentation broth was added, the inhibition effect was basically as high as 87.65%.

Claims

1. A trichosporon, It is characterized in that It was named Trichosporon sp., strain number was ZJU2023TR1, and the preservation number was CCTCC NO: M 20231367.

2. Use of the Trichosporon according to claim 1 in the preparation of a plant pathogen inhibitor, wherein the plant pathogen is Magnaporthe oryzae or Cochliobolus heterostrophus.

3. A plant pathogen inhibitor, It is characterized in that The active ingredient comprises the Trichosporon according to claim 1 or the fermentation liquid of the Trichosporon according to claim 1.

4. The plant pathogen inhibitor according to claim 3, It is characterized in that The volume percentage concentration of the fermentation liquid of Trichosporon is 10% to 40%.

5. A method for preparing the plant pathogen inhibitor according to claim 3 or 4, It is characterized in that When the active ingredient of the plant pathogen inhibitor comprises the fermentation broth of the Trichosporon according to claim 1, the preparation method is: The plant pathogen inhibitor is prepared by inoculating the trichosporon described in claim 1 into a fermentation medium for cultivation and removing the bacterial bodies from the fermentation liquid.

6. The method for preparing the plant pathogen inhibitor according to claim 5, It is characterized in that The culture temperature was 25°C and the culture period was 4 days.

7. Use of the trichosporon according to claim 1 or the plant pathogen inhibitor according to claim 3 or 4 in inhibiting the growth of plant pathogens, wherein the plant pathogen is rice blast fungus or corn leaf blight pathogen.

8. A method for preventing and controlling plant pathogens, It is characterized in that The trichosporon described in claim 1 or the plant pathogen inhibitor described in claim 3 or 4 is sprayed on the plants.

9. The method for controlling plant pathogens according to claim 8, It is characterized in that The plant is rice or corn.

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