Metarhizium anisopliae DY2-9 and application thereof in preventing and treating plant diseases

Abstract

The invention relates to metarhizium anisopliae DY2-9 and application thereof in preventing and treating plant diseases. The Metarhizium anisopliae sp. DY2-9 is preserved in the China General Microbiological Culture Collection Center on March 28, 2025, the preservation address is No.3, No.1 Yard, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 41857. The Metarhizium anisopliae sp. DY2-9 has the advantages that the Metarhizium anisopliae sp. The invention also provides a culture method of the strain and application of the strain in preparation of a fungicide for preventing and treating plant pathogenic fungal diseases. The metarhizium anisopliae DY2-9 with remarkable antagonistic activity on plant pathogenic fungi is screened, the strain has remarkable bacteriostatic action on 14 plant pathogenic fungi, the inhibition rate can reach 45% or above, and the metarhizium anisopliae DY2-9 not only has an extremely good broad-spectrum bacteriostatic function, but also has an extremely strong bacteriostatic effect, has great application potential in biological prevention and control of plant diseases, and can be widely applied to biological prevention and control of plant diseases. The method can reduce the use of chemical pesticides, reduce pesticide residues in agricultural products, protect the environment, and meet the urgent demand on the production of pollution-free agricultural products at present.

Description

Technical Field

[0001] The invention relates to a strain of Metarhizium anisopliae DY2-9 and application thereof in preventing and controlling plant diseases, belonging to the technical field of biological prevention and control of plant diseases. Background Art

[0002] Ensuring global food security is one of the major challenges facing humanity in the 21st century. According to the Food and Agriculture Organization of the United Nations (FAO), the global population is projected to reach 9.7 billion by 2050. To meet the demands of this growing population and evolving dietary patterns, food production will need to increase by 50-110% from current levels. Plant diseases, particularly fungal diseases, are a core threat to crop health and high grain yields. Globally, direct economic losses from plant diseases reach $40 billion annually, with 70-80% of known plant diseases caused by fungal pathogens. Notably, these pathogens not only reduce crop yields but also produce mycotoxins (such as aflatoxins and vomitoxin) that threaten human health through the food chain. Of particular concern is the over-sevenfold increase in the detection rate of newly identified plant pathogens since 2000. This surge is closely linked to the monoculture model of intensive agriculture. Long-term selective pressure from chemical pesticides has driven adaptive evolution in pathogen populations, leading to the spread of drug-resistant genotypes. Establishing a multi-target prevention and control system has become a top priority. The current mainstream plant pathogen and fungal prevention and control strategies include chemical control, resistance breeding and biological control.

[0003] While chemical control methods have a historic role in plant disease management, overreliance on chemical pesticides has led to the emergence of resistant pathogens, toxicity to humans and animals, and environmental pollution. To address the shortcomings of chemical pesticides, the development of biopesticides with excellent antimicrobial activity plays a key role in agricultural production.

[0004] Biocontrol fungi possess unique advantages in ecological adaptability, possessing robust secondary metabolite synthesis capabilities and producing a variety of antimicrobial compounds, including terpenes and polyketides. Crucially, biocontrol fungi share overlapping ecological niches with pathogens, enabling sustained pest control through mechanisms such as antagonism, hyperparasitism, nutrient competition, and spatial occupancy. Research has revealed that over 300 strains from at least 113 fungal genera (such as Trichoderma, Aspergillus, and Penicillium) have been identified as possessing biocontrol potential. Among these, Trichoderma has achieved the most successful industrial application, with approximately 60% of commercial biofungicides worldwide derived from strains of this genus (such as Trichoderma harzianum). These fungi exhibit remarkable effectiveness in controlling soil-borne diseases such as damping-off and gray mold through synergistic effects, including the secretion of chitinases to degrade pathogen cell walls, the secretion of antibacterial compounds to inhibit pathogens, and the activation of plant defense responses.

[0005] Filamentous fungi hold great potential for controlling plant pathogens. While studies have identified specific types of filamentous fungi with inhibitory activity against plant pathogens, their activity is often unstable, susceptible to physical and chemical factors, and their antagonistic activity varies. Continuing to identify novel biocontrol fungi with strong antagonistic abilities will promote the development of new biopesticides and ensure the sustainable development of green agriculture in the future. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides a strain of Metarhizium anisopliae DY2-9 and its application in preventing and controlling plant diseases.

[0007] The technical solutions of the present invention are as follows: A strain of Metarhizium anisopliae ( Metarhizium sp. )DY2-9, the strain was deposited in the General Microbiology Center of China Culture Collection Administration on March 28, 2025, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.41857.

[0008] According to the present invention, preferably, the Metarhizium anisopliae ( Metarhizium sp. ) The ITS gene sequence of DY2-9 is shown in SEQ ID NO.1.

[0009] The above-mentioned method for culturing Metarhizium anisopliae DY2-9 comprises the following steps: The Metarhizium DY2-9 was inoculated on a PDA solid culture medium and cultured at 25-30°C for 5-10 days to obtain Metarhizium DY2-9 spores; the Metarhizium DY2-9 spores were then inoculated into a PDB liquid culture medium, cultured at 25-30°C and 180-220 rpm for 2-5 days, and then transferred to a fresh PDB liquid culture medium and continued for 5-10 days to obtain a Metarhizium DY2-9 fermentation broth.

[0010] The use of the above-mentioned Metarhizium DY2-9 or Metarhizium DY2-9 fermentation liquid in the preparation of fungal agents for preventing and controlling plant pathogenic fungal diseases.

[0011] According to the present invention, preferably, the plant pathogenic fungi include nut fruit rot pathogens ( Lasiodiplodia pseudotheobroma ), Fusarium graminearum ( Fusarium gramineae )、Mango deformity pathogen ( Fusarium mango trees ), banana wilt pathogen ( Fusarium oxysporum f. sp. cubense )、 Fusarium stinking 、 Fusarium oxysporum f. sp. phaseoli 、Tomato early blight pathogen ( Alternaria nightshade ), Tung oil tree blight pathogen ( Nectria sp.)、Alternaria ( Alternaria alternata ), Botrytis cinerea ( Botrytis cinerea ) 、Corynebacterium maydis( Cochliobolus heterostrophus ) 、Fusarium rotundifolia( Fusarium verticillium ) 、Fusarium oxysporum( Fusarium oxysporum )、Peach brown rot fungus( Monilinia fructifera ) one or more.

[0012] A bacterial agent for preventing and treating plant diseases, comprising the above-mentioned Metarhizium anisopliae DY2-9 or Metarhizium anisopliae DY2-9 fermentation liquid.

[0013] Where the present invention is not exhaustive, existing technologies may be used.

[0014] Beneficial effects 1. The present invention screened out Metarhizium anisopliae ( Metarhizium sp. )DY2-9, which is effective against nut fruit rot pathogens ( Lasiodiplodia pseudotheobromae ), Fusarium graminearum ( Fusarium gramineae )、Mango deformity pathogen ( Fusarium mangosteen ), banana wilt pathogen ( Fusarium oxysporum f. sp. cubense )、 Fusarium wilt 、 Fusarium oxysporum f. bean sp. 、Tomato early blight pathogen ( Alternaria solani ), Tung oil tree blight pathogen ( Nectria sp. )、Alternaria ( Alternaria alternata ), Botrytis cinerea ( Botrytis cinerea ) 、Corynebacterium maydis( Cochliobolus heterostrophus ) 、Fusarium rotundifolia( Fusarium verticillium ) 、Fusarium oxysporum( Fusarium oxysporum )、Peach brown rot fungus( Monilinia fructifera ) and other 14 plant pathogenic fungi, and the inhibition rate can reach more than 45%. B. cinerea ), Bipolaris maydis ( C. heterostrophus ) and peach brown rot pathogen ( M. fructicola ) were all over 90%, and the inhibition rate against Alternaria ( A. alternata ) and tung blight pathogen ( Nectria sp. ) with an inhibition rate of over 80%. It not only has excellent broad-spectrum antibacterial function, but also has a strong antibacterial effect, and has great application potential in the biological control of plant diseases.

[0015] 2. The Metarhizium DY2-9 or Metarhizium DY2-9 fermentation liquid of the present invention may have application prospects in preparing fungal agents for preventing and controlling plant pathogenic fungal diseases, and is expected to provide a new biocontrol agent for the prevention and control of plant pathogenic fungal diseases, which can reduce the use of chemical pesticides, reduce pesticide residues in agricultural products, and protect the environment, meeting the current urgent demand for the production of pollution-free agricultural products. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 This is the result of screening and identification of Metarhizium anisopliae DY2-9; In the figure, a shows the confrontation culture of Metarhizium DY2-9 against Fusarium rotundifolia; b shows the growth of Metarhizium DY2-9 on PDA solid culture medium; c shows the molecular biological identification results of Metarhizium DY2-9.

[0016] Figure 2 This is the result of the effect of Metarhizium anisopliae DY2-9 on the hyphal morphology of Fusarium rotundifolia; In the figure, a is the mycelial morphology of Fusarium rotundifolia cultured alone; b is the mycelial morphology of Fusarium rotundifolia cultured in confrontation with Metarhizium anisum DY2-9.

[0017] Figure 3 This is the physicochemical stability test result of the antibacterial activity of Metarhizium anisopliae DY2-9; In the figure, a is the temperature tolerance test result; b is the pH tolerance test result; c is the ultraviolet radiation stability test result; d is the metal ion tolerance test result.

[0018] Figure 4 The inhibitory effect of Metarhizium anisopliae DY2-9 fermentation liquid on plant pathogenic fungi and Aspergillus fumigatus; Among them, + represents the growth status of the pathogen on the PDA medium supplemented with the fermentation broth of Metarhizium anisopliae DY2-9; - represents the growth status of the pathogen on the PDA medium.

[0019] Figure 5 This is the inhibitory effect of the fermentation broth of Metarhizium DY2-9 on bacteria and yeast.

[0020] Figure 6 The effect of Metarhizium anisopliae DY2-9 fermentation liquid on inhibiting Botrytis cinerea infection in tomato leaves; In the figure, a is the result of treatment 1; b is the result of treatment 2; + represents soaking in the fermentation liquid of Metarhizium anisopliae DY2-9 for 30 min; - represents soaking in sterile water for 30 min. DETAILED DESCRIPTION

[0021] The technical solution of the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto.

[0022] The culture and formulation used in the embodiments of the present invention are as follows: PDA medium: 200 g peeled potatoes, 20 g glucose, 1000 mL distilled water; 15 g agarose (solid medium).

[0023] PDB liquid medium: 200 g peeled potatoes, 20 g glucose, 1000 mL distilled water.

[0024] Nut fruit rot pathogens used in the examples ( Lasiodiplodia pseudotheobromae ), Fusarium graminearum ( Fusarium gramineae )、Mango Anthracnose ( Colletotrichum asianum )、Mango deformity pathogen ( Fusarium mangosteen ), banana wilt pathogen ( Fusarium oxysporum f. sp. from Cuba )、 Fusarium wilt 、 Fusarium oxysporum f. sp. phaseoli 、Tomato early blight pathogen ( Alternaria solani ), Tung oil tree blight pathogen ( Nectria sp. )、Alternaria ( Alternaria alternate )、Botrytis cinerea( Botrytis cinerea ) 、Corynebacterium maydis( Snail heterostrophe ) 、Fusarium rotundifolia( Fusarium verticillioides ) 、Fusarium oxysporum( Fusarium oxysporum )、Peach brown rot fungus( Monilinia fructicola )、Pepper Anthracnose ( Colletotrichum capsicum )、Aspergillus fumigatus( Aspergillus fumigatus )、Aflatoxin( Aspergillus flavus ) are all conventional strains preserved by the applicant and can also be purchased directly from the preservation center.

[0025] Example 1. Isolation and identification of strain DY2-9 1. A total of 23 soil samples were collected from different habitats across the country, and fungi were isolated, resulting in a total of 395 soil fungi strains.

[0026] 2. Antagonistic activity against Fusarium verticillata (FV) and mycoparasitism was preliminarily evaluated against 395 isolated fungal strains using the plate standoff method. Thirty-six fungal strains demonstrated varying degrees of biocontrol activity against FV, categorized by mechanism of action as either antagonistic or mycoparasitic. Twenty-eight strains demonstrated significant antagonistic activity, representing 7.09% of the total tested strains.

[0027] Among these 28 fungal strains, the most noteworthy one was the strain labeled DY2-9, which showed the most obvious antagonistic activity by forming an obvious inhibition zone. The specific results are as follows: 图1 As shown in Fig. a, the antibacterial activity of the DY2-9 strain in confrontation experiment was 51.34%.

[0028] 3. The DY2-9 strain was inoculated on PDA solid medium, and after 7 days of culture at 28°C, the colony diameter reached 4.0 cm, white aerial hyphae were formed on the periphery, and yellow conidia appeared in the center; after 14 days of culture, the colony expanded to 7.28 cm and produced a large number of yellow conidia, and the back of the colony was characteristic yellow-brown. The specific morphology is shown in Fig. b. 图1

[0029] 4. The DY2-9 strain was inoculated into a conical flask containing 10 mL of PDB liquid medium, and cultured at 28°C with shaking at 180 rpm for 1-2 days, then centrifuged at 8000 rpm for 10 min to collect the bacterial cells. The genomic DNA was extracted according to the instructions of the OMEGA fungal genomic DNA extraction kit. The ITS, RPB1, RPB2 and TEF1 genes of the DY2-9 strain were obtained by PCR amplification using the extracted genomic DNA as the template and the sequences in Table 1 as the primers.

[0030] Table 1 The PCR amplification conditions were as follows: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 1 min, annealing at 55°C for 1 min, extension at 72°C for 90 s, and post-extension at 72°C for 5 min, for 30 cycles.

[0031] The PCR products were recovered and purified according to the instructions of the OMEGA gel recovery kit. The PCR products were sent to Qingdao Qikao Biotechnology Co., Ltd. for sequencing analysis. According to the sequence information of the ITS gene (as shown in SEQ ID NO. 1), the RPB1 gene (as shown in SEQ ID NO. 2), the RPB2 gene (as shown in SEQ ID NO. 3) and the TEF1 gene (as shown in SEQ ID NO. 4) of the DY2-9 strain, the BLAST tool of NCBI was used to perform similarity analysis with the sequences of the reported typical strains. The results showed that it had the highest similarity value with Metarhizium, and it was determined that it was very likely a Metarhizium strain.

[0032] The genomic DNA of the DY2-9 strain was sent to Huada Biotechnology Co., Ltd. for whole genome sequencing. The published whole genome sequence of Metarhizium was downloaded from the Genome database in NCBI, and the MEGA 7 software was used for whole genome phylogenetic tree analysis. The results are shown in Fig. c. 图1

[0033] As shown in Fig. c. 图1 ​​c It can be seen that DY2-9 forms an independent evolutionary branch within the genus Metarhizium, and is most closely related to Metarhizium robertsii and Metarhizium anisopliae.

[0034] 4. The morphological response characteristics of Fusarium rotundifolia hyphae cultured alone and cultured in opposition with DY2-9 were compared and analyzed by scanning electron microscopy (SEM). 图2 shown.

[0035] Depend on 图2 It can be seen that the hyphae of the Fusarium rotundifolia colony cultured opposite DY2-9 curled and deformed, and the DY2-9 strain can significantly destroy the cell structure integrity of the pathogen. Microscopic observation showed that the hyphae of Fusarium rotundifolia cultured alone showed typical filamentous fungal morphological characteristics, with uniform hyphae diameter, smooth and homogeneous surface, intact cell wall-membrane structure, and uniform cytoplasm distribution ( 图2 a); The strain cultured against DY2-9 showed obvious structural damage, with abnormal swelling of the hyphae tips and severe shrinkage in local areas, accompanied by cell wall collapse and membrane rupture, forming obvious cytoplasm leakage channels ( 图2 b).

[0036] Based on the above results, the inventors of this application successfully screened out a strain with significant antagonistic effect on Fusarium rotundifolia and named it Metarhizium anisopliae ( Metarhizium sp. )DY2-9.

[0037] Finally, Metarhizium anisopliae ( Metarhizium sp. ) DY2-9 is deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number being CGMCC No. 41857.

[0038] Example 2, Metarhizium anisopliae ( Metarhizium sp. ) Physical and chemical stability test of antibacterial substances in DY2-9 fermentation broth 1. A method for culturing Metarhizium anisopliae DY2-9, comprising the following steps: The green anisopliae DY2-9 isolated in Example 1 was inoculated on PDA solid culture medium and cultured at 28°C for 7 days to obtain green anisopliae DY2-9 spores; then the green anisopliae DY2-9 spores were inoculated into 250 mL of PDB liquid culture medium, cultured at 28°C and 200 rpm for 3 days, and then transferred to 250 mL of fresh PDB liquid culture medium and continued for 6 days. After filtering with a G1 funnel, the green anisopliae DY2-9 fermentation liquid was obtained.

[0039] 2. The stability of the antibacterial active substances in the fermentation broth of Metarhizium anisopliae DY2-9 was evaluated by temperature tolerance experiment. The specific method is as follows: The fermentation broth of Metarhizium anisopliae DY2-9 was divided into four equal volumes and treated in a water bath at 37°C, 50°C, 75°C, and 100°C for 30 minutes, respectively. The mixture was then allowed to stand at room temperature for 24 hours to obtain the heat-treated fermentation broth. The heat-treated fermentation broth was then added to sterilized PDA solid medium (equilibrated in a 50°C water bath) at a ratio of 1:10 (v / v) to form the experimental group (Dt). Untreated fermentation broth of Metarhizium anisopliae DY2-9 served as the positive control (CK), and an equal volume of sterile water was used instead of the fermentation broth as the negative control (Dc).

[0040] An 8 mm diameter Fusarium verticillata (FV) cake was inoculated in the center of the experimental group, positive control group, and negative control group, and cultured at 28°C. Three replicates were set up for each group, and growth dynamics were observed daily. When the negative control FV colony was close to full, the colony diameters of the experimental group (Dt), positive control group (CK), and negative control group (Dc) were measured, and the inhibition rate was calculated: I = (Dc / CK-Dt) / Dc × 100%. The results are shown in the figure. 图3 As shown in a.

[0041] Depend on 图3 aIt can be seen that the fermentation broth of Metarhizium DY2-9 still maintains an inhibition rate of 70.15% after heat treatment at 100°C, which is only 2.13 percentage points lower than the inhibition rate of 72.28% of the positive control group (CK), and still maintains 97% activity (p>0.05), confirming that its active substances have excellent thermal stability. The active substances in the fermentation broth of Metarhizium DY2-9 may be heat-resistant small molecule secondary metabolites. This discovery provides an important basis for the subsequent separation and purification strategy of active ingredients.

[0042] 3. The pH tolerance test system was used to evaluate the acid-base stability of the antibacterial active substance of Metarhizium anisopliae DY2-9. The specific method is as follows: The fermentation broth of Metarhizium anisopliae DY2-9 was aliquoted into seven equal volumes. The pH of each aliquot was adjusted to 1.0, 3.0, 5.0, 7.0, 9.0, 11.0, and 13.0 using 1 mol / L NaOH and HCl solutions, respectively. After standing at room temperature for 24 hours, the pH of the fermentation broth was adjusted back to its original pH using the same concentrations of acid and alkali. This neutralized fermentation broth was then added to sterilized PDA solid medium (equilibrated in a 50°C water bath) at a ratio of 1:10 (v / v) to form the experimental group (Dt). Untreated fermentation broth of Metarhizium anisopliae DY2-9 served as the positive control (CK), and an equal volume of sterile water was used instead of the fermentation broth of Metarhizium anisopliae DY2-9 as the negative control (Dc).

[0043] An 8 mm diameter Fusarium verticillata (FV) cake was inoculated in the center of the experimental group, positive control group, and negative control group, and cultured at 28°C. Three replicates were set up for each group, and growth dynamics were observed daily. When the negative control FV colony was close to full, the colony diameters of the experimental group (Dt), positive control group (CK), and negative control group (Dc) were measured, and the inhibition rate was calculated: I = (Dc / CK-Dt) / Dc × 100%. The results are shown in the figure. 图3 As shown in b.

[0044] Depend on 图3 b It can be seen that the fermentation broth of Metarhizium DY2-9 showed excellent acid and alkali tolerance: except for the inhibition rate in the pH 13.0 treatment group dropped to 46.75% (a decrease of 35.7% compared with the inhibition rate of 72.28% in the positive control group, p < 0.05), the inhibition rates after the remaining pH gradient (1.0-11.0) treatments were maintained above 62.34% (p > 0.05), and the activity remained above 85%. The active substances in the fermentation broth of Metarhizium DY2-9 have a broad spectrum of pH stability.

[0045] 4. The UV irradiation stability test system was used to evaluate the UV tolerance of the antibacterial active substance acid of Metarhizium anisopliae DY2-9. The specific method is as follows: Equal volumes of Metarhizium DY2-9 fermentation broth were aliquoted into four aliquots and irradiated under 254 nm UV light (radiation intensity 0.5 W / m²) for 2, 4, 6, and 8 hours, respectively, to obtain irradiated Metarhizium DY2-9 fermentation broth. The irradiated Metarhizium DY2-9 fermentation broth was then added to sterilized PDA solid medium (equilibrated in a 50°C water bath) at a ratio of 1:10 (v / v) to form the experimental group (Dt). Untreated Metarhizium DY2-9 fermentation broth served as the positive control (CK), and an equal volume of sterile water was used in place of Metarhizium DY2-9 fermentation broth as the negative control (Dc).

[0046] An 8 mm diameter Fusarium verticillata (FV) cake was inoculated in the center of the experimental group, positive control group, and negative control group, and cultured at 28°C. Three replicates were set up for each group, and growth dynamics were observed daily. When the negative control FV colony was close to full, the colony diameters of the experimental group (Dt), positive control group (CK), and negative control group (Dc) were measured, and the inhibition rate was calculated: I = (Dc / CK-Dt) / Dc × 100%. The results are shown in the figure. 图3 As shown in c.

[0047] Depend on 图3 As shown in Figure 3, a gradient of UV exposure time (0 / 2 / 4 / 6 / 8 h) had no significant effect on the antibacterial activity of the M. anisopliae DY2-9 fermentation broth (p>0.05). Quantitative analysis showed that the antibacterial rate of the M. anisopliae DY2-9 fermentation broth remained stable above 69.05%, confirming the excellent UV stability of the active substances in the M. anisopliae DY2-9 fermentation broth.

[0048] 5. The ion stability characteristics of the fermentation broth of Metarhizium anisopliae DY2-9 were evaluated by the metal ion tolerance test system. The specific method is as follows: The fermentation broth of Metarhizium anisopliae DY2-9 was divided into four equal volumes. NaCl, KCl, CaCl2, CuSO4, MgCl2, and FeCl3 solutions (all at a final concentration of 0.01 M) were then added, respectively, and the mixture was allowed to stand at room temperature for 24 hours to obtain the metal ion-treated fermentation broth of Metarhizium anisopliae DY2-9. The metal ion-treated fermentation broth of Metarhizium anisopliae DY2-9 was then added to sterilized PDA solid medium (equilibrated in a 50°C water bath) at a ratio of 1:10 (v / v) to form the experimental group (Dt). Untreated fermentation broth of Metarhizium anisopliae DY2-9 served as the positive control (CK), and an equal volume of sterile water was used in place of the Metarhizium anisopliae DY2-9 fermentation broth as the negative control (Dc).

[0049] An 8 mm diameter Fusarium verticillata (FV) cake was inoculated in the center of the experimental group, positive control group, and negative control group, and cultured at 28°C. Three replicates were set up for each group, and growth dynamics were observed daily. When the negative control FV colony was close to full, the colony diameters of the experimental group (Dt), positive control group (CK), and negative control group (Dc) were measured, and the inhibition rate was calculated: I = (Dc / CK-Dt) / Dc × 100%. The results are shown in the figure. 图3 As shown in d.

[0050] Depend on 图3 d shows that the antibacterial activity of the fermentation broth of Metarhizium anisopliae DY2-9 showed a relatively stable tolerance to metal ions, but Ca 2+ (65.99%) and Cu 2+ (34.57%) treatment groups resulted in a decrease in the inhibition rate, among which Cu 2+ The inhibitory effect was the most significant, with a decrease of 52.5% (p < 0.01); + (87.99%) and Mg 2+ The (81.68%) treatment groups produced a synergistic effect, with the antibacterial effects increased by 21.0% (p < 0.01) and 12.3% (p < 0.05), respectively.

[0051] Example 3: Determination of the antibacterial effect of Metarhizium anisopliae DY2-9 on plant pathogenic fungi The antibacterial effect of Metarhizium anisopliae DY2-9 on plant pathogenic fungi was determined as follows: (1) Nut rot pathogens ( Lasiodiplodia pseudotheobromae ), Fusarium graminearum ( Fusarium graminearum )、Mango Anthracnose ( Colletotrichum asianum )、Mango deformity pathogen ( Fusarium mangiferae ), banana wilt pathogen ( Fusarium oxysporum f. sp. cubense )、 Fusarium foetens 、 Fusarium oxysporum f. sp. phaseoli 、Tomato early blight pathogen ( Alternaria solani ), Tung oil tree blight pathogen ( Nectria sp. )、Alternaria ( Alternaria alternata ), Botrytis cinerea ( Botrytis cinerea ) 、Corynebacterium maydis( Cochliobolus heterostrophus ) 、Fusarium rotundifolia( Fusarium verticillioides ) 、Fusarium oxysporum( Fusarium oxysporum )、Peach brown rot fungus( Monilinia fructicola )、Pepper Anthracnose ( Colletotrichum capsicum )、Aspergillus fumigatus( Aspergillus fumigatus )、Aflatoxin( Aspergillus flavus ) were inoculated on PDA solid culture medium and cultured at 28°C for 2-3 days to obtain 18 plant pathogenic fungi; (2) The Metarhizium DY2-9 isolated in Example 1 was inoculated on a PDA solid culture medium and cultured at 28°C for 7 days to obtain Metarhizium DY2-9 spores; then the Metarhizium DY2-9 spores were inoculated into 250 mL of PDB liquid culture medium, cultured at 28°C and 200 rpm for 3 days, and then transferred to 250 mL of fresh PDB liquid culture medium for another 6 days. After filtering with a G1 funnel, a fermentation broth of Metarhizium DY2-9 was obtained; (3) After filtering the DY2-9 fermentation broth of Metarhizium anisopliae through a 0.22 μm sterile filter membrane, add it to the sterilized PDA medium (50°C water bath insulation) at a ratio of 1:10 (v / v), and then add it to the sterilized PDA solid medium (50°C water bath equilibrium) at a ratio of 1:10 (v / v) as the experimental group (Dt); at the same time, replace the DY2-9 fermentation broth of Metarhizium anisopliae with an equal volume of sterile water as the control group (Dc); then inoculate the pathogenic fungus cake (Φ8 mm) cultured in step (1) in the center of the experimental group and the control group respectively, and culture at a constant temperature of 28°C. Set up 3 replicates for each group, and observe the growth dynamics every day. When the colony of the control group is close to full, measure the colony diameters of the experimental group (Dt) and the control group (Dc), and calculate the antibacterial rate: I = (Dc -Dt) / Dc×100%. The results are as follows: 图4 and as shown in Table 2.

[0052] Table 2. Inhibitory effect of the fermentation liquid of Metarhizium anisopliae DY2-9 on plant pathogenic fungi and Aspergillus fumigatus Depend on 图4 As shown in Table 2, the fermentation liquid of Metarhizium anisopliae DY2-9 is effective against mango anthracnose pathogen ( Colletotrichum asianum )、Pepper Anthracnose ( Colletotrichum capsicum )、Aspergillus fumigatus( Aspergillus fumigatus ) and Aspergillus flavus ( Aspergillus flavus ) had no significant inhibitory effect on the other nut fruit rot pathogens ( Lasiodiplodia pseudotheobromae ), Fusarium graminearum ( Fusarium graminearum )、Mango deformity pathogen ( Fusarium mangiferae ), banana wilt pathogen ( Fusarium oxysporum f. sp. cubense )、 Fusarium foetens 、 Fusarium oxysporum f. sp. phaseoli 、Tomato early blight pathogen ( Alternaria solani ), Tung oil tree blight pathogen ( Nectria sp. )、Alternaria ( Alternaria alternata ), Botrytis cinerea ( Botrytis cinerea ) 、Corynebacterium maydis( Cochliobolus heterostrophus ) 、Fusarium rotundifolia( Fusarium verticillioides ) 、Fusarium oxysporum( Fusarium oxysporum )、Peach brown rot fungus( Monilinia fructicola ) and other 14 plant pathogenic fungi showed significant antagonistic effects.

[0053] Further analysis revealed that the fermentation liquid of Metarhizium anisopliae DY2-9 was effective against Botrytis cinerea ( Botrytis cinerea ) 、Corynebacterium maydis( Cochliobolus heterostrophus ) and peach brown rot pathogen ( Monilinia fructicola ) were all over 90%; Alternaria alternata ) and tung blight pathogen ( Nectria sp. ) with an inhibition rate of over 80%, indicating that Metarhizium anisum DY2-9 not only has excellent broad-spectrum antibacterial function, but also has a strong antibacterial effect, and has great application potential in the biological control of plant diseases.

[0054] The above data results show that Metarhizium DY2-9 can synthesize compounds with broad-spectrum antifungal activity, and Metarhizium DY2-9 or its fermentation liquid shows important application value in the field of biological control of plant pathogenic fungi.

[0055] Example 4 The sensitivity of Metarhizium anisopliae DY2-9 fermentation broth to Staphylococcus aureus ( S. aureus ), Pseudomonas aeruginosa ( P.aeruginosa ), Escherichia coli ( E. coli ), Bacillus subtilis ( B. subtilis ) and Saccharomyces cerevisiae ( S.cerevisiae ) and Candida albicans ( C.albicans ), the specific method is as follows: (1) Saccharomyces cerevisiae ( S.cerevisiae ) and Candida albicans ( C.albicans ) were inoculated into YPD liquid medium and cultured overnight at 30℃ and 200 rpm in a shaking incubator until OD600nm=2.0; Staphylococcus aureus ( S. aureus ), Pseudomonas aeruginosa ( P.aeruginosa ), Escherichia coli ( E. coli ) and Bacillus subtilis ( B. subtilis ) were inoculated into LB liquid medium and cultured overnight at 37°C and 200 rpm in a shaking incubator until OD600nm = 2.0; the culture medium of the above strains was then centrifuged to remove the medium, and an equal volume of fresh YPD liquid medium or LB liquid medium was added and set aside to obtain pathogenic bacterial cultures of two fungi and four bacteria; (2) The Metarhizium DY2-9 isolated in Example 1 was inoculated on a PDA solid culture medium and cultured at 28°C for 7 days to obtain Metarhizium DY2-9 spores; the Metarhizium DY2-9 spores were then inoculated into 250 mL of PDB liquid culture medium, cultured at 28°C and 200 rpm for 3 days, and then transferred to 250 mL of fresh PDB liquid culture medium for 6 days. After filtering with a G1 funnel, a Metarhizium DY2-9 fermentation liquid was obtained; the Metarhizium DY2-9 fermentation liquid was then freeze-dried and concentrated to prepare concentrated Metarhizium DY2-9 fermentation liquids with concentrations of 2 times, 5 times, and 10 times the original concentration; Amphotericin B and streptomycin were prepared with sterile water to obtain 4 mg / mL amphotericin B solution and streptomycin solution, respectively. (3) Add 400 μL of the pathogenic bacteria liquid obtained in step (1) to 250 mL of YPD solid medium or LB solid medium, mix well and pour into a plate, place a 6 mm diameter round drug-sensitive sheet on the surface of the solidified medium, and take 10 μL of Metarhizium DY2-9 fermentation liquid and concentrated Metarhizium DY2-9 fermentation liquid and drop them on the center of the drug-sensitive sheet respectively, evaporate to dryness, and use them as experimental groups (1×, 2×, 5×, 10×); at the same time, add equal volumes of DMSO (fungus) and sterile water (bacteria) as the negative control group, and add equal volumes of amphotericin B solution (fungus) and streptomycin solution (bacteria) as the positive control group. Culture in a constant temperature incubator for 24 h, observe the size of the inhibition zone, and the results are as follows: Figure 5 shown.

[0056] Depend on Figure 5 The results show that when concentrated at 2×, 5×, and 10×, the fermentation broth of Metarhizium anisopliae DY2-9 showed no inhibitory activity against either Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) or Gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). However, when concentrated to 10×, the fermentation broth significantly inhibited the growth of Saccharomyces cerevisiae and Candida albicans. This suggests that the antimicrobial activity of the fermentation broth of Metarhizium anisopliae DY2-9 is highly selective, with its antibacterial components acting specifically against some pathogenic fungi while having no significant inhibitory effect against prokaryotic microorganisms.

[0057] Example 5 The detached leaf infection experiment was used to evaluate the antimicrobial activity of Metarhizium anisopliae DY2-9 fermentation broth against Botrytis cinerea ( Botrytis cinerea ) to achieve the prevention and control effect, the specific methods are as follows: (1) Five-week-old healthy leaves from tomato seedlings at the same position and maturity were taken and treated in two groups. The first group was immersed in sterile water for 30 min, the liquid was evaporated, and the leaves were placed in sterile PDA solid medium as the control group. The second group was immersed in the fermentation liquid of Metarhizium anisopliae DY2-9 for 30 min, the liquid was evaporated, and the leaves were placed in sterile PDA solid medium as the experimental group. Three replicates were set for each of the two groups. (2) Inoculate Botrytis cinerea on PDA solid culture medium and culture at 28°C for 2-3 days to obtain Botrytis cinerea spores and Botrytis cinerea blocks; then drop 150 μL of Botrytis cinerea spores (concentration of 1.01×10 7 CFU / mL), each treatment was repeated 3 times, cultured at 28℃ for 5 days under dark conditions, and the size of the bacterial plaque was observed and recorded, which was used as treatment 1; Botrytis cinerea was inoculated at the same position of the leaves by the point grafting method, and each treatment was repeated 3 times, cultured at 28℃ for 5 days under dark conditions, and the size of the bacterial plaque was observed and recorded, which was used as treatment 2; the results of the two treatments are shown in Figure 2. Figure 6 shown.

[0058] Depend on Figure 6 It can be seen that in 150 μL of Botrytis cinerea spore suspension (1.01×10 7 CFU / mL) inoculated leaves, the control group formed irregular elliptical lesions with a diameter of 2.6-3.4 cm in the radial direction and 1.2-1.7 cm in the horizontal direction, while the treated group only retained unevaporated spore liquid ( Figure 6 a, Treatment 1). When inoculated with Botrytis cinerea, the lesions in the control group expanded into a nearly circular shape with a diameter of 1.4-1.6 cm, while the treatment group only formed a dark inhibition zone at the edge of the lesion, with no substantial tissue infection ( Figure 6 b, treatment 2). This indicates that the fermentation liquid of Metarhizium anisopliae DY2-9 can effectively block the infection process of Botrytis cinerea on tomato leaves.

[0059] The above-described embodiments are only preferred specific implementation plans of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A strain of Metarhizium anisopliae ( Metarhizium sp. ) DY2-9, characterized in that The strain was deposited in the General Microbiology Center of China Culture Collection Administration on March 28, 2025. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No. 41857.

2. The Metarhizium anisopliae DY2-9 according to claim 1, characterized in that The ITS gene sequence of the Metarhizium anisopliae DY2-9 is shown in SEQ ID NO.

1.

3. The method for culturing Metarhizium anisopliae DY2-9 according to claim 1, characterized in that: The steps are as follows: The green anisopliae DY2-9 described in claim 1 is inoculated on a PDA solid culture medium and cultured at 25-30°C for 5-10 days to obtain green anisopliae DY2-9 spores; then the green anisopliae DY2-9 spores are inoculated into a PDB liquid culture medium, cultured at 25-30°C and 180-220rpm for 2-5 days, and then transferred to a fresh PDB liquid culture medium and continued for 5-10 days to obtain a green anisopliae DY2-9 fermentation liquid.

4. Use of the Metarhizium anisopliae DY2-9 according to claim 1 or the fermentation liquid of Metarhizium anisopliae DY2-9 according to claim 3 in the preparation of fungal agents for preventing and controlling plant pathogenic fungal diseases.

5. The use according to claim 4, characterized in that The plant pathogenic fungi include nut fruit rot pathogens ( Lasiodiplodia pseudotheobromae ), Fusarium graminearum ( Fusarium graminearum )、Mango deformity pathogen ( Fusarium mangiferae ), banana wilt pathogen ( Fusarium oxysporum f. sp. cubense )、 Fusarium foetens 、 Fusarium oxysporum f. sp. phaseoli 、Tomato early blight pathogen ( Alternaria solani ), Tung oil tree blight pathogen ( Nectria sp. )、Alternaria ( Alternaria alternata ), Botrytis cinerea ( Botrytis cinerea ), Bipolaris maydis ( Cochliobolus heterostrophus )、Fusarium rotundifolia( Fusarium verticillioides ), Fusarium oxysporum ( Fusarium oxysporum )、Peach brown rot fungus( Monilinia fructicola ) one or more.

6. A fungal agent for preventing and controlling plant diseases, characterized in that: The bacterial agent includes the Metarhizium anisopliae DY2-9 described in claim 1 or the Metarhizium anisopliae DY2-9 fermentation broth described in claim 3.

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