A kind of trichosporon and its application

Abstract

This invention discloses a pyrrosporon sp. and its applications, relating to the field of plant pathogen control technology. The pyrrosporon sp. is named *Trichosporon* sp., strain number ZJU2023TR1, and its preservation number is CCTCC NO: M 20231367. In plate confrontation experiments, the pyrrosporon sp. ZJU2023TR1 provided by this invention can inhibit the growth of *Trichosporon oryzae* and *Trichosporon melilotus* pathogens; in fermentation broth experiments, the inhibition rate against *Trichosporon oryzae* is as high as 87.65%, and the inhibition rate against *Trichosporon melilotus* pathogens is as high as 91.58%. This indicates that strain ZJU2023TR1 can effectively inhibit the growth of *Trichosporon oryzae* and *Trichosporon melilotus* pathogens, and can be used to prepare novel, highly efficient, and broad-spectrum biocontrol agents, providing a new approach for the development of microbial applications in biological control.

Description

Technical Field

[0001] This invention relates to the field of plant pathogen control technology, and in particular to a type of filamentous yeast and its application. Background Art

[0002] Rice blast, also known as rice fever, is one of the most important diseases affecting rice. The severity of this disease varies greatly depending on the variety, cultivation management, and climatic conditions. In years with widespread rice blast, yield reductions can generally reach 10%–20%, and in severe years, reductions can reach 40%–50%, or even result in complete crop failure in some fields. Rice blast can occur throughout the entire growth cycle of rice and can be classified into seedling blast, leaf blast, neck blast, and grain blast based on the affected part of the rice plant. The pathogen of rice blast is *Magnaportheoryzae*, which is characterized by its wide host range, easy mutation leading to new physiological races, and strong resistance in its conidia and mycelium. This has made rice blast surpass sheath blight and bacterial blight as the most prevalent of the three major rice diseases. The main measures for controlling rice blast disease include selecting superior 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 need to continuously innovate within the existing rice blast disease prevention and control system, constantly explore new potential genes and green pesticides, and thus ensure high and stable rice yields and maximize its social benefits.

[0003] Maize leaf spot is a significant disease affecting maize production. It is widely distributed and prevalent, with some areas experiencing more severe outbreaks, severely impacting maize yields. In some warm and humid regions of my country, such as the Huang-Huai-Hai summer maize area, leaf spot is widespread and severe in some areas. The pathogen is *Cochliobolusheterostrophus*, which primarily affects leaves, but leaf sheaths, husks, and ears can also be infected. This fungus can occur throughout the entire maize growth cycle, but is most severe during the tasseling and grain-filling stages. In warm and humid areas of the Yellow River and Yangtze River basins in my country, maize leaf spot is widespread and severe, generally causing yield losses of 15-20%, with severe cases resulting in losses exceeding 50%, or even total crop failure. It is generally more severe in summer maize areas, and in years with large-scale outbreaks, significant yield losses can occur.

[0004] Biological control refers to the method of controlling plant diseases and pests using organisms and their metabolic products. Its essence is to regulate the population density of harmful organisms by utilizing interspecific and intraspecific relationships, i.e., controlling one group of organisms with another. Compared with traditional control techniques, biological control has advantages such as not polluting the environment, being safe for humans and other organisms, leaving no residues, being easily coordinated with other plant protection measures, and saving energy. It plays an increasingly important role in integrated pest management. To date, fungi, bacteria, actinomycetes, and yeasts can all be used for biological control.

[0005] In actual production processes, traditional pesticide control is prevalent. However, according to literature reports, pesticide utilization rates are generally only 10%, with approximately 90% of pesticides remaining in the environment, causing pollution. Furthermore, pesticide residues can also harm humans and animals. Therefore, screening for 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* sp. strain and its application. The strain ZJU2023TR1 was isolated from soil at the Zijingang Campus of Zhejiang University, Hangzhou, Zhejiang Province (30.312106°N, 120.097711°E). Sequence comparison showed that the ZJU2023TR1 strain is most closely related to the *Trichosporon* sp. sequence, indicating that the isolated ZJU2023TR1 strain is a *Trichosporon* sp. strain.

[0007] This invention provides a fungus named Trichosporon sp., strain number ZJU2023TR1, with accession number CCTCC NO: M 20231367.

[0008] The present invention also provides the application of the aforementioned mycelium in the preparation of plant pathogen inhibitors, wherein the plant pathogen is Magnaphalthe oryzae or Cochliobolusheterostrophus.

[0009] The present invention also provides a plant pathogen inhibitor, the active ingredient of which includes the aforementioned Mycosaccharomyces cerevisiae or the fermentation broth of the aforementioned Mycosaccharomyces cerevisiae.

[0010] When strain ZJU2023TR1 was cultured in confrontation with the pathogens of maize leaf spot and rice blast, the mycelial growth inhibition rates of the two pathogens reached 25.62% and 24.62%, respectively. When cultured on fermentation broth containing strain ZJU2023TR1, the inhibitory effect on maize leaf spot and rice blast was significant.

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

[0012] When the plant pathogen is the corn leaf spot pathogen, the volume percentage concentration of the fermentation broth of the soy yeast is 10% to 30%. When cultured on a culture medium with this volume percentage concentration of fermentation broth, the inhibition effect against the corn leaf spot pathogen is as high as 66.71% when 10% fermentation broth is added, and the inhibition effect against the corn leaf spot pathogen is as high as 91.58% when 30% fermentation broth is added.

[0013] When the plant pathogen is rice blast fungus, the volume percentage concentration of the fermentation broth of the soy yeast is 10% to 40%. When cultured on a culture medium with this volume percentage concentration of fermentation broth, the inhibition effect on rice blast fungus is as high as 12.55% when 10% fermentation broth is added, and the inhibition effect on rice blast fungus is as high as 87.65% when 40% fermentation broth is added.

[0014] This invention also provides a method for preparing the plant pathogen inhibitor, wherein the active ingredient of the plant pathogen inhibitor includes the fermentation broth of the *Mycosaccharomyces cerevisiae*, and the preparation method is as follows:

[0015] After inoculating the aforementioned *Mycosaccharomyces cerevisiae* into a fermentation medium and culturing it, the fermentation broth after removing the bacterial cells is the plant pathogen inhibitor. The culture temperature is 25℃, and the culture time is 4 days.

[0016] The present invention also provides the application of the aforementioned *Mycospora* or the aforementioned plant pathogen inhibitor in inhibiting the growth of plant pathogens, wherein the plant pathogen is *Oryza sativa* or *Oryza sativa*.

[0017] This invention also provides a method for controlling plant pathogens, which involves spraying the plant with the aforementioned *Mycospora* or the aforementioned plant pathogen inhibitor. The plant is rice or corn.

[0018] The beneficial effects of this invention are:

[0019] The *Zygosaccharomyces cerevisiae* strain ZJU2023TR1 provided by this invention can inhibit the growth of *Oryza sativa* and *Spodoptera exigua* in plate confrontation tests; in fermentation broth tests, the inhibition rate against *Oryza sativa* is as high as 87.65%, and the inhibition rate against *Spodoptera exigua* is as high as 91.58%. This indicates that strain ZJU2023TR1 can effectively inhibit the growth of *Oryza sativa* and *Spodoptera exigua*, and can be used to prepare novel, highly efficient, and broad-spectrum biocontrol agents, providing a new approach for the development of microbial applications in biological control. Attached Figure Description

[0020] Figure 1The images show the colony morphology of *Saccharomyces cerevisiae* ZJU2023TR1 after 8 days of growth on PDA medium; where A is the front view of the culture dish and B is the back view of the culture dish.

[0021] Figure 2 This is a diagram of the hyphal morphology of *Saccharomyces cerevisiae* ZJU2023TR1.

[0022] Figure 3 The graph shows the results of the inhibition of corn leaf spot pathogen by *Mycosaccharomyces cerevisiae* ZJU2023TR1; where A represents the inhibition of corn leaf spot pathogen by *Mycosaccharomyces cerevisiae* ZJU2023TR1, and B represents the growth status of corn leaf spot pathogen.

[0023] Figure 4 The graph shows the results of the inhibition of rice blast fungus ZJU2023TR1 by *Mycospermum oryzae*. In the graph, A shows the inhibition of rice blast fungus by *Mycospermum oryzae* ZJU2023TR1, and B shows the growth status of rice blast fungus.

[0024] Figure 5 The graphs and bar charts show the inhibitory effect of the fermentation broth of *Mycosaccharomyces cerevisiae* ZJU2023TR1 on the pathogen of corn leaf blight. A represents the inhibitory effect of the fermentation broth of *Mycosaccharomyces cerevisiae* ZJU2023TR1 on the pathogen of corn leaf blight, and B represents the bar chart showing the inhibitory effect of the fermentation broth of *Mycosaccharomyces cerevisiae* ZJU2023TR1 on the pathogen of corn leaf blight.

[0025] Figure 6 The graphs and bar charts show the inhibitory effect of the fermentation broth of *Mycosaccharomyces cerevisiae* ZJU2023TR1 on *Bacillus oryzae*. A represents the inhibitory effect of the fermentation broth of *Mycosaccharomyces cerevisiae* ZJU2023TR1 on *Bacillus oryzae*, and B represents the bar chart showing the inhibitory effect of the fermentation broth of *Mycosaccharomyces cerevisiae* ZJU2023TR1 on *Bacillus oryzae*. Detailed Implementation

[0026] Example 1: Isolation of bacterial strains

[0027] (1) Soil samples were collected from the mulberry garden of Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang Province. The selective medium dilution plate method was used for separation. The procedure is as follows: Take four 1.5 mL centrifuge tubes and mark them with a marker. -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 1g of sample and place it in a stoppered test tube containing 9mL of sterile water. Shake for 10min to obtain 10g of sample. -1 Soil dilution solution;

[0029] (3)10 -1 After shaking and allowing the soil dilution to stand for 2 minutes, pipette 0.1 mL of the suspension into a centrifuge tube containing 0.9 mL of sterile water. Dilute sequentially to prepare 10... -2 10 -3 10 -4 10 -5 10 -6 Diluent;

[0030] (4) Pour 20 mL of sterilized PDA medium (with streptomycin and ampicillin added to each medium to reach 30 μg / L to inhibit bacterial growth) cooled to 50-60℃ into petri dishes; after the medium has cooled to form plates, use a pipette to aspirate 10 mL of the medium into each petri dish. -6 10 -5 10 -4 10 -3 Add 0.1 mL of the diluent suspension to a plate, and then spread each diluent evenly with a sterile spreader. Repeat 3 plates for each concentration.

[0031] (5) Invert the petri dish and incubate it in a constant temperature incubator at 25℃ for 3-5 days. Then pick a single colony of mycelium and place it on a new plate.

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

[0033] A strain was isolated from the soil of Zhejiang University Zijingang Campus in Hangzhou, Zhejiang Province (30.312106°N, 120.097711°E) and named ZJU2023TR1.

[0034] Example 2: Identification of fungal strain morphology

[0035] After 8 days of dark cultivation on PDA medium at 25°C, the colony diameter ranged from 36.32 to 42.64 mm, with an average mycelial growth rate of 4.54 to 5.33 mm per day. It took 13.13 to 15.44 days to completely cover a 70 mm diameter plate. The colonies exhibited an irregular, outward-spreading pattern, sometimes forming several smaller, irregularly shaped colonies of varying sizes. The upper surface of the colonies was generally cheese-white. Figure 1 A), the back is pale yellow with an orange tinge ( Figure 1B). Colonies near the center of the front side exhibit a porous, wrinkled, and gyrate morphology, while those further away from the center gradually become smoother and extend irregularly outwards; colonies on the back side are smooth and lack any distinctive morphology. No conidia of ZJU2023TR1 were observed under a 100x microscope, but its dense and abundant hyphae were visible. The hyphae are slender and uniform, slightly pointed at the ends, and no hyphal septa were observed under a 100x microscope. Figure 2 ).

[0036] Based on the combined characteristics of colony morphology, hyphae morphology, and molecular biological identification, strain ZJU2023TR1 was identified as a mycelial yeast.

[0037] Example 3: Molecular Identification

[0038] (1) DNA extraction

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

[0040] The extraction buffer formulation is: 10 mM ethylenediaminetetraacetic acid, 100 mM Tris-hydrochloric acid, 1 M potassium chloride;

[0041] 2) Place the centrifuge tubes in MP In a -24°C homogenizer, the mycelial tissue was broken by oscillation at 65 Hz for 2 minutes.

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

[0043] 4) Add an equal volume of isopropanol to the supernatant, invert the tube to mix well, and place the centrifuge tube in a 4°C freezer for 10 minutes to precipitate DNA better.

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

[0045] 6) Add 800 μL of 70% ethanol (v / v) to dissolve the impurities, mix gently, and centrifuge at 12000 rpm for 5 min.

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

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

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

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

[0050] PCR amplification was performed in a 20 μL reaction system containing: 1 μL each of forward and reverse primers, 10 μL of Green Taq Mix, 2 μL of template DNA, and 6 μL of ddH2O.

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

[0052] (3) Gene sequencing and sequence analysis

[0053] The target DNA fragment was sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing. After rigorous verification, the sequencing results yielded DNA fragment sequences as shown in SEQ ID No. 1 and SEQ ID No. 2, namely 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 and the 28S ribosomal large subunit rRNA gene (LSU) of strain ZJU2023TR1 were searched and compared with homologous or similar nucleotide sequences in the GenBank database using BLAST. Sequence comparison showed that strain ZJU2023TR1 was most closely related to the sequence of *Trichosporon* sp., a result consistent with the morphological identification, indicating that the isolated strain ZJU2023TR1 is a *Trichosporon* sp. This newly screened strain was named *Trichosporon* sp., with strain number ZJU2023TR1, and was deposited on July 24, 2023, at the China Center for Type Culture Collection (CCTCC), Wuhan University, China, with accession number CCTCC NO: M 20231367.

[0055] Example 4: Inhibitory effect of strain ZJU2023TR1 on the pathogen of maize leaf spot disease

[0056] The ZJU2023TR1 strain was subjected to confrontation culture with the pathogen of maize leaf spot: On a 90 mm diameter PDA plate, the pathogen of maize leaf spot and the ZJU2023TR1 strain were inoculated on opposite sides of the plate, 5 cm apart. After 7 days of confrontation culture on the PDA plate, the growth of the colony leading edge of the maize leaf spot pathogen was inhibited in the direction closer to the ZJU2023TR1 strain, and the mycelium showed a significant inhibition state. Figure 3 According to the measurements and calculations, the mycelial growth inhibition rate can reach 25.62% (growth inhibition rate = (colon radius of corn leaf spot pathogen - colony radius of corn leaf spot pathogen in confrontation culture) / colony radius of corn leaf spot pathogen).

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

[0058] The ZJU2023TR1 strain was subjected to confrontation culture with *Oryza sativa*: *Oryza sativa* and *B. sativa* strain ZJU2023TR1 were inoculated onto opposite sides of a 90 mm diameter PDA plate, 4.5 cm apart. After 8 days of confrontation culture on the PDA plate, the growth of the *Oryza sativa* colony leading edge closer to *B. sativa* strain ZJU2023TR1* was inhibited, and the mycelium showed significant inhibition. Figure 4 According to the measurements and calculations, the mycelial growth inhibition rate can reach 24.62% (growth inhibition rate = (rice blast fungus colony radius - rice blast fungus colony radius in confrontation culture) / rice blast fungus colony radius).

[0059] Example 6: Detection of the inhibitory effect of fermentation broth of strain ZJU2023TR1 on the pathogen of maize leaf spot disease.

[0060] The ZJU2023TR1 strain was inoculated into 100 mL PDA medium / 250 mL Erlenmeyer flask and cultured at 25℃ and 150 rpm for 4 days. The supernatant was collected by centrifugation and used as the fermentation broth. After filtration and sterilization, it was added to PDA medium in a specific ratio to prepare fermentation broth-PDA plates. These plates were then inoculated with the corn leaf blight pathogen, and the antibacterial effect was observed after 4 days. The corn leaf blight pathogen was inoculated into PDA medium without fermentation broth, and with 10%, 20%, and 30% fermentation broth (v / m) respectively, and cultured for 4 days. Three replicates were set up for each group at different concentrations. Figure 5 As shown in Figure A, the higher the concentration of the fermentation broth, the more significant the inhibitory effect on the pathogen of corn leaf blight. The inhibitory effect is as follows: Figure 5 As shown in bar chart B, the inhibition rate reached 66.71% when 10% fermentation broth was added. When 30% fermentation broth was added, the inhibition rate reached approximately 91.58%.

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

[0062] ZJU2023TR1 strain was inoculated into 150 mL PDA medium / 250 mL Erlenmeyer flask and cultured at 25℃ and 150 rpm for 4 days. The supernatant was collected by centrifugation and used as the fermentation broth. After filtration and sterilization, it was added to CM medium in a specific ratio to prepare fermentation broth-CM plates. These plates were then inoculated with rice blast fungus, and the antibacterial effect was observed after 4 days. Rice blast fungus was inoculated onto CM medium containing no fermentation broth, and containing 10%, 20%, 30%, and 40% fermentation broth (v / v) and cultured for 4 days. Three replicates were set up for each group at different concentrations. Figure 6 ).like Figure 6 As shown in Figure A, the higher the concentration of fermentation broth, the more significant the inhibitory effect on rice blast fungus, while the control group showed no inhibitory effect on rice blast fungus. The relative inhibition rates are shown in Figure A. Figure 6 As shown in Figure B, the inhibition rate was as high as 12.55% when 10% fermentation broth was added. When 40% fermentation broth was added, the inhibition rate reached approximately 87.65%.

Claims

1. A type of filamentous yeast, characterized in that, Named Trichosporon sp., Plant No. ZJU2023TR1, Preservation No. CCTCC NO: M 20231367.

2. The use of the *Mycospora* strain according to claim 1 in the preparation of plant pathogen inhibitors, wherein the plant pathogen is *Pseudomonas oryzae* (rice blast fungus). Magnaporthe oryzae ) or the pathogen of corn leaf blight ( Cochliobolus heterostrophus ).

3. A plant pathogen inhibitor, characterized in that, The active ingredient includes the *Mycosaccharomyces cerevisiae* as described in claim 1 or the fermentation broth of the *Mycosaccharomyces cerevisiae* as described in claim 1; The plant pathogen is rice blast fungus ( ). Magnaporthe oryzae ) or the pathogen of corn leaf blight ( Cochliobolus heterostrophus ).

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

5. The method for preparing the plant pathogen inhibitor according to claim 3 or 4, characterized in that, When the active ingredient of the plant pathogen inhibitor includes the fermentation broth of the *Saccharomyces cerevisiae* as described in claim 1, the preparation method is as follows: After the *Mycosaccharomyces cerevisiae* of claim 1 is inoculated into a fermentation medium and cultured, the fermentation broth after removing the bacterial cells is used as a plant pathogen inhibitor.

6. The method for preparing the plant pathogen inhibitor according to claim 5, characterized in that, The culture temperature was 25ºC, and the culture time was 4 days.

7. The application of the *Mycospora* fungus of claim 1, or the plant pathogen inhibitor of claim 3 or 4, in inhibiting the growth of plant pathogens, wherein the plant pathogen is *Pseudomonas oryzae* (rice blast fungus). Magnaporthe oryzae ) or the pathogen of corn leaf blight ( Cochliobolus heterostrophus ).

8. A method for controlling plant pathogens, characterized in that, The plant pathogen inhibitor described in claim 1 or claim 3 or 4 is sprayed onto the plant. The plant pathogen is rice blast fungus ( ). Magnaporthe oryzae ) or the pathogen of corn leaf blight ( Cochliobolus heterostrophus ); The plant is either rice or corn.

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