A strain of goniomonas viridis pe-1 and its application

Abstract

The application discloses a Gliocladium catenatum PE-1, which is classified as Gliocladium catenatum Clonostachys rosea f. catenulata Clonostachys rosea f , is preserved in the China General Microbiological Culture Collection Center on February 2, 2024, and has a preservation number of CGMCC NO. 41164. The application also discloses a bacterial agent prepared from the Gliocladium catenatum PE-1. The application further discloses applications of the Gliocladium catenatum PE-1 and the bacterial agent in preventing and treating plant diseases. The Gliocladium catenatum PE-1 screened in the application can widely induce the burst of active oxygen of a plant immune marker in monocotyledonous plants and dicotyledonous plants, and can effectively prevent and treat various plant diseases, such as soybean phytophthora root rot, rice rice blast, wheat powdery mildew and wheat scab, and provides a new strain resource and technical support for biological control of plant diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microorganism technology, and in particular to a strain of Sporosarcina ureae PE-1 and application thereof. BACKGROUND

[0002] At present, the main methods for preventing and controlling plant diseases are planting disease-resistant varieties and using chemical agents. Planting disease-resistant varieties is the most economical, safe and effective measure for preventing and controlling plant diseases in agricultural production. However, due to the long breeding cycle of disease-resistant varieties, the lack of disease-resistant resources for some crops, the multiple types of pathogenic fungi, the complex pathogenic groups and the fast mutation speed, the planting of disease-resistant varieties is still limited. Using chemical agents for prevention and control is the most widely used prevention and control measure, which is convenient to operate and has good prevention and control effect, but the chemical agent residues caused by long-term application of chemical agents may threaten food safety and soil ecological environment. Therefore, the use of environmentally friendly and sustainable biological control strategies has a bright development prospect.

[0003] Soil microorganisms are important biocontrol resource banks, and a large number of biocontrol agents have been discovered and isolated from soil by researchers. The use of biocontrol agents for biological control can effectively reduce the use of chemical agents. Biocontrol agents can control the occurrence of diseases through competition, antagonism, parasitism, induction of plant resistance and promotion of plant growth. This multiple mode of action makes the disease control more stable and sustainable, and has good ecological protection function. At the same time, biocontrol agents isolated from the original niche of soil may also have more long-acting control potential. Therefore, further exploration and application of biocontrol agents is an effective measure for preventing and controlling plant diseases. SUMMARY

[0004] The present application aims to provide a strain of Sporosarcina ureae PE-1 and application thereof, so as to solve the problems in the prior art.

[0005] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0006] The present application provides a strain of Sporosarcina ureae PE-1 in the first aspect. Clonostachys rosea f. catenulata The Sporosarcina ureae PE-1 is preserved in the China General Microbiological Culture Collection Center, and the preservation date is February 2, 2024, and the preservation number is CGMCC NO.41164.

[0007] The present application provides a microbial agent prepared from the above-mentioned Sporosarcina ureae PE-1 in the second aspect.

[0008] Further, the bacterial agent is prepared by the following method: inoculating the Gliocladium cymbiforme PE-1 to a PDA culture medium plate, culturing at 25-28℃ in dark for 12-14d, cutting the Gliocladium cymbiforme PE-1 plate after culturing into small pieces, putting the Gliocladium cymbiforme PE-1 plate small pieces into MM liquid medium, culturing at 25-28℃ in dark with 160-180rpm for 5-7d to obtain a fermentation broth; filtering the fermentation broth with multi-layer gauze, and then filtering the obtained filtrate with a filter membrane to obtain a fermentation supernatant, freezing and drying the fermentation supernatant to obtain Gliocladium cymbiforme PE-1 fermentation broth freeze-dried powder, and the bacterial agent is prepared by using sterile water, wherein the bacterial agent contains 1-10mg / mL of the Gliocladium cymbiforme PE-1 fermentation broth freeze-dried powder.

[0009] Further, the filter membrane is a 0.45μm filter membrane.

[0010] The third aspect of the present application provides application of the above-mentioned Gliocladium cymbiforme PE-1 in preventing and treating plant diseases.

[0011] Further, the plant includes monocotyledon, dicotyledon; the monocotyledon includes Poaceae, and the dicotyledon includes Solanaceae, Leguminosae, Cruciferae, Musaceae and Malvaceae; the Poaceae includes wheat, corn and rice, the Solanaceae includes potato and tomato, the Leguminosae includes soybean, the Cruciferae includes Chinese cabbage, the Musaceae includes banana, and the Malvaceae includes cotton.

[0012] Further, the plant disease includes soybean Phytophthora root rot, rice blast, wheat powdery mildew and wheat scab.

[0013] The fourth aspect of the present application provides application of the above-mentioned bacterial agent in preventing and treating plant diseases.

[0014] Further, the plant includes monocotyledon, dicotyledon; the monocotyledon includes Poaceae, and the dicotyledon includes Solanaceae, Leguminosae, Cruciferae, Musaceae and Malvaceae; the Poaceae includes wheat, corn and rice, the Solanaceae includes potato and tomato, the Leguminosae includes soybean, the Cruciferae includes Chinese cabbage, the Musaceae includes banana, and the Malvaceae includes cotton.

[0015] Further, the plant disease includes soybean Phytophthora root rot, rice blast, wheat powdery mildew and wheat scab.

[0016] The present application has the following advantages:

[0017] This invention screened a strain of Polysporus emblica PE-1, whose fermentation broth can widely induce the release of reactive oxygen species, a plant immune marker, in both monocotyledonous and dicotyledonous plants. It can effectively prevent and control a variety of plant diseases, including soybean root rot, rice blast, wheat powdery mildew, and wheat scab. It provides new strain resources and technical support for the biological control of plant diseases.

[0018] Unlike traditional fungicides, *Aspergillus emblica* PE-1 exhibits a broad-spectrum immune-inducing effect on plants, making it a promising candidate for application against various plant diseases. *Aspergillus emblica* PE-1 can significantly enhance plant disease resistance and reduce disease incidence, including against soybean root rot caused by *Phytophthora infestans*, rice blast, wheat powdery mildew, and wheat scab. *Aspergillus emblica* PE-1 is highly effective in controlling plant diseases, environmentally friendly, and avoids the persistent pollution caused by chemical pesticides, thus possessing extremely high development and utilization value. Attached Figure Description

[0019] Figure 1 This is a curve showing the reactive oxygen species (ROS) emission from the roots of soybean etiolated seedlings induced by the fermentation broth of strain PE-1. RLU represents fluorescence intensity.

[0020] Figure 2 This is a colony photograph of strain PE-1.

[0021] Figure 3 Phylogenetic tree of strain PE-1.

[0022] Figure 4 This is a graph showing the reactive oxygen species (ROS) emission curves induced by the fermentation broth of *Alternaria pelargoniflora* PE-1 in Example 3 for different types of plants. RLU represents fluorescence intensity.

[0023] Figure 5 The control effect of Polyporus emblica PE-1 fermentation broth on soybean Phytophthora root rot in Example 4 is shown.

[0024] Figure 6 The control effect of Polyporus emblica PE-1 fermentation broth on rice blast disease in Example 4 is shown.

[0025] Figure 7 The control effect of Polyporus emblica PE-1 fermentation broth on wheat powdery mildew in Example 4 is shown.

[0026] Figure 8 The control effect of the fermentation broth of Polyporus emblica PE-1 in Example 4 on wheat scab. Information on the preservation of biological materials

[0027] Strain PE-1, classified as *Alternaria emblica*. Clonostachys rosea f. catenulataIt is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, on February 2, 2024, with accession number CGMCC NO.41164. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. The following embodiments are intended to facilitate a better understanding of the present invention, but do not limit the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0030] The culture media involved in the following examples are as follows:

[0031] MM liquid culture medium: glucose 10g, sodium nitrate 6g, potassium chloride 0.52g, magnesium sulfate heptahydrate 0.312g, dipotassium hydrogen phosphate 1.52g, vitamin B1 0.01g, 1mL 1000X trace element solution, and deionized water to a final volume of 1L. Sterilize at 121℃ for 20min.

[0032] 1000X Trace Element Solution: 22g magnesium sulfate heptahydrate, 11g boric acid, 5g manganese chloride tetrahydrate, 1.7g cobalt chloride hexahydrate, 1g copper sulfate pentahydrate, 1.5g sodium manganate dihydrate, 50g tetrasodium EDTA, and deionized water to a final volume of 1L. Store at 4℃.

[0033] PDA medium: Cut 200g of peeled potatoes into small pieces. Add 800mL of deionized water to a pot, bring to a boil, then add the potatoes and cook for 15-20 minutes until the liquid becomes viscous. Add 20g of glucose to a beaker, place two to three layers of gauze at the mouth of the beaker, and pour the viscous potato liquid from the gauze into the beaker. Then, bring the volume to 1L with deionized water and add 20g of agar powder. Sterilize at 121℃ for 20 minutes.

[0034] V8 medium: Add 1g CaCO3 to 100mL V8 juice, mix well on a magnetic stirrer for 10min, centrifuge at 3000rpm for 5-7min at room temperature, take the supernatant, make up to 1L with deionized water, and add 15g agar powder. Sterilize at 121℃ for 20min.

[0035] SDC medium: 100g (dry weight) rice straw (about 1cm long), add appropriate amount of deionized water, boil for 20min, filter with two layers of gauze, discard the residue, add 40g corn flour to the filtrate, then make up to 1L with deionized water, add 15g agar powder. Sterilize at 121℃ for 20min.

[0036] Unless otherwise specified, the plates (petition dishes) used in the following examples all have a diameter of 90 mm.

[0037] Example 1 Isolation and Identification of Strain PE-1

[0038] 1. Isolation of strain PE-1

[0039] 1.1 Obtaining test materials

[0040] Healthy, well-grown soybean plants were selected from soybean fields in Lishui District, Nanjing City, Jiangsu Province, and their root tissues were collected.

[0041] 1.2 Treatment of soybean root tissue

[0042] Multiple soybean root tissue blocks, approximately 2 mm × 2 mm in size, were cut. The soybean root tissue blocks were sequentially immersed in 75 v / v ethanol for 30 seconds, then in 2 m / v sodium hypochlorite solution for 2 minutes, and finally rinsed three times in sterile water. The soybean root tissue blocks were then blotted dry with sterile absorbent paper. Three to four soybean root tissue blocks were placed on PDA agar plates containing 50 μg / mL rifampin and 50 μg / mL ampicillin, and incubated at 25°C in the dark for 1–2 days. If colonies appeared on the soybean root tissue blocks, they were immediately transferred to fresh PDA agar plates and incubated at 25°C in the dark.

[0043] 1.3 Activity Screening

[0044] The isolated strain (fungus, 5mm mycelial cake) was inoculated onto plates containing 15mL of PDA medium and cultured at 25℃ in the dark for 6-7 days. The cultured plates were then cut into approximately 5mm × 5mm pieces (strain plate pieces) using a sterile scalpel and placed in MM liquid medium (2 plates of strain plate pieces per 1.5L MM liquid medium). Fermentation was carried out in a shaker for 7 days at 25℃ in the dark at 160rpm. The resulting fermentation broth was filtered through two layers of gauze, and the filtrate was then filtered through a 0.45μm filter membrane to obtain the fermentation supernatant. The fermentation supernatant was used to detect reactive oxygen species in the roots of soybean etiolated seedlings.

[0045] Fill cylindrical pots (32cm inner diameter, 35cm inner height) with 20cm of sterile vermiculite, moisten the vermiculite with sterile water, and sow 50 soybeans (Hefeng 47 variety; vermiculite height above the soybeans approximately 1cm) in each pot. Grow the soybeans in darkness at 25℃ and 70% relative humidity for 3-4 days to obtain yellow soybean seedlings (the above-ground height of the yellow soybean seedlings is approximately 5cm). Select 12 yellow soybean seedlings with relatively uniform growth from each strain.

[0046] 1 cm of the root tip was cut off from soybean etiolated seedlings, and 8 cm of the root tip removed was used as experimental material. The soybean etiolated seedling roots were then cut into 5 mm segments, and two segments were randomly selected and placed in one well of a 96-well plate. 200 µL of sterile water was added to each well. The 96-well plate was placed in an incubator at 25 °C in the dark for 30 min. Afterward, all the sterile water in each well was aspirated, and 100 µL of reactive oxygen species reaction solution was added to form the treatment group (PE-1); 100 µL of MM liquid culture medium was added to form the control group (CK). The reactive oxygen species (ROS) reaction solution (200 µL system) included horseradish peroxidase (P6782-10 mg, SIGMA), common luminol (8511-5 G, SIGMA), fermentation supernatant of the strain, and sterile water. The final concentration of horseradish peroxidase was 10 µg / mL (1000 times diluted stock solution, prepared with sterile water), the final concentration of common luminol was 35.4 µg / mL (1000 times diluted stock solution, prepared with DMSO), the fermentation supernatant of the strain was 100 µL, and the remainder was sterile water. Immediately after adding the ROS reaction solution, the 96-well plate was placed in a GLOMAX96 microplate spectrophotometer (Promega, Madison, WI, USA) to detect the ROS bursting in soybean etiolated seedling roots. Light protection was maintained during the experiment. This led to the screening of strain PE-1, which can promote the ROS bursting in soybean etiolated seedling roots and activate the immune response in soybean. Figure 1 ).

[0047] 2. Identification of strain PE-1

[0048] 2.1 Morphological identification

[0049] Strain PE-1 was cultured on PDA medium at 25°C in the dark. Figure 2 As shown, the colonies are initially white, gradually turning light green at the center as they mature, while the edges remain white. The hyphae are cottony or fluffy, with a moist or slightly dry surface (depending on the culture medium). When conidia are produced in large quantities, the colony surface exhibits a powdery or granular structure (due to spore aggregation).

[0050] 2.2 Molecular biological identification

[0051] 2.2.1 Genomic DNA of strain PE-1 was extracted using a kit (DP320-50) from Tiangen Biotech (Beijing) Co., Ltd.

[0052] 2.2.2 Amplification of genomic DNA of strain PE-1 EF1 -a, BenA(β-tubulin) gene. The DNA amplification reaction system was 25 μL, including 12.5 μL of 2×PCR Mix, 1 μL each of forward and reverse primers (10 μM) (primer sequences are shown in Table 1), 2 μL of template DNA, and 8.5 μL of ddH2O. The PCR amplification conditions were as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, for a total of 34 cycles; and a final extension at 72℃ for 10 min.

[0053] 2.2.3 Sequencing Results: Sequencing results showed that strain PE-1... EF1 -a, BenA The gene sequences are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively.

[0054] 2.2.4 The obtained gene sequences were compared on the NCBI database website, and a phylogenetic tree was constructed. Figure 3 ).

[0055] Therefore, strain PE-1 is presumed to be *Acetobacter emblica*. Clonostachys rosea f. catenulata ( C. rosea f. catenulata It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.41164.

[0056] Table 1

[0057]

[0058] Example 2: Preparation of freeze-dried powder from fermentation broth of Polyporus emblica PE-1

[0059] Fresh, activated *Agropyron emblica* PE-1 mycelia (5mm mycelial cakes) were picked and inoculated onto plates containing 15mL of PDA medium. The plates were then incubated at 25℃ in the dark for 14 days. Using a sterile scalpel, the incubated *Agropyron emblica* PE-1 plates were cut into approximately 5mm × 5mm pieces (*Agropyron emblica* PE-1 plate pieces) and placed in MM liquid medium (2 plates of *Agropyron emblica* PE-1 plate pieces per 1.5L MM liquid medium). Fermentation was carried out at 25℃ in the dark at 160rpm for 7 days to obtain the fermentation broth. The fermentation broth was filtered through two layers of gauze, and the filtrate was then filtered through a 0.45μm filter membrane to obtain the fermentation supernatant. The fermentation supernatant was freeze-dried (at -30℃ and 3-5Pa for 48h) to obtain *Agropyron emblica* PE-1 fermentation broth freeze-dried powder.

[0060] Example 3: Broad-spectrum induction of plant immunity (reactive oxygen species burst) in monocotyledonous and dicotyledonous plants by fermentation broth of Polysporus emblica PE-1.

[0061] Reactive oxygen species (ROS) bursts are important markers of plant immune induction. In this example, *Polyspora pellucida* PE-1 fermentation broth was used. Fresh, intact leaves from monocotyledonous plants (wheat, corn, rice) and dicotyledonous plants (potato, tomato, soybean, cabbage, banana, cotton) were selected. Leaf discs with a diameter of 2.5 mm were punched from the upper surface of the leaves using a punch, or the leaves were cut into 2.5 mm × 2.5 mm squares using a blade. These leaf discs were placed in 96-well microplates with 200 μL of sterile water added to each well, ensuring the underside of the leaves was facing upwards. After punching the leaf discs, the entire 96-well microplate was incubated overnight at room temperature in the dark. On the second day, all sterile water in each well of the 96-well microplate was gently aspirated with a sterile pipette tip. It is crucial to remove all water completely to avoid affecting subsequent treatments. The following experimental results were obtained: A mixture containing regular luminol, horseradish peroxidase, and lyophilized powder of Polyporus umbellatus PE-1 fermentation broth was prepared using sterile water. The final concentration of regular luminol was 35.4 μg / mL (the stock solution was 1000 times diluted and prepared with DMSO), the final concentration of horseradish peroxidase was 10 µg / mL (the stock solution was 1000 times diluted and prepared with sterile water), and the final concentration of lyophilized powder of Polyporus umbellatus PE-1 fermentation broth was 8 mg / mL. This mixture was added to the above 96-well microplate, 200 μL per well, as the experimental group (PE-1). Then, it was placed in a GLOMAX96 microplate spectrophotometer (Promega, Madison, WI, USA) to measure its luminescence value. Light protection was required during the experiment. A control group (CK) was set up for each plant, which was a mixture containing a fixed volume of luminol (35.4 μg / mL) and horseradish peroxidase (10 µg / mL) instead of the mixture containing luminol (35.4 μg / mL), horseradish peroxidase (10 µg / mL), and freeze-dried Polysporus emblica PE-1 fermentation broth (8 mg / mL). Each plant experimental group and control group had 12 replicates.

[0062] like Figure 4 The results showed that the fermentation broth of Polyporus emblica PE-1 can widely induce the release of reactive oxygen species, a plant immune marker, in both monocots and dicots, and has the potential to become a broad-spectrum inducing agent for important crops including those in the Poaceae, Solanaceae, Fabaceae, Brassicaceae, Musaceae, and Malvaceae families.

[0063] Example 4: Effect of Polyporus emblica PE-1 fermentation broth on plant disease control

[0064] 1. Effect of Polyporus emblica PE-1 fermentation broth on control of Phytophthora root rot in soybean:

[0065] 1.1 Highly virulent strains of Phytophthora soybean Phytophthora sojaeJS2 (i.e. PsJS2; see reference 1: Li Ke, Zheng Sujiao, Wang Xiaoli, Sun Zhe, Ye Wenwu, Wang Yuanchao, Zheng Xiaobo. Evaluation of resistance of 251 soybean varieties (lines) to Phytophthora sojae and various Fusarium species [J]. Journal of Nanjing Agricultural University, 2022, 45(2): 261-268. Reference 2: Yang Jin, Wang Xiaoman, Ye Wenwu, Zheng Xiaobo, Wang Yuanchao. Identification of resistance of soybean germplasm resources to Phytophthora root rot in the Huang-Huai-Hai region [J]. Soybean Science, 2020, 39(1): 012-022.) was inoculated onto 15 mL V8 medium plates and cultured at 25℃ in the dark for 7 days for later use.

[0066] 1.2 The round pots used for soybean pot cultivation had an inner diameter of 15cm and an inner height of 13cm. The pots were filled with sterile vermiculite. The soybean variety was Williams. Each pot contained 15 soybean seeds (with vermiculite about 1cm above the seeds). The pots were then placed in a greenhouse for 10 days. Greenhouse conditions included: temperature 25℃, light (3500K, 30000LUX) for 16 hours, darkness for 8 hours, and relative humidity of 50%. The vermiculite was thoroughly watered before planting the soybean seeds, and then watered every 3 days, using approximately 200mL of sterile water per pot each time.

[0067] 1.3 Ten days after planting, the experimental group (PE-1) received 100 mL of *Polyporus emblica* PE-1 fermentation broth (8 mg / mL freeze-dried *Polyporus emblica* PE-1 fermentation broth, prepared with sterile water) at the base of each soybean seedling. The control group (CK) received the same volume of sterile water. Both the experimental and control groups were replicated in four groups. Twenty-four hours later, wound inoculation was performed on the soybean seedling stem (1.5 cm below the cotyledons) by making a 1 cm long longitudinal incision on the stem using a sterile blade; a hole punch was then used in step 1.1 of this embodiment. Phytophthora sojae Print 5mm diameter mycelium cakes on the JS2 plate and place the mycelium cakes on the wounds of soybean seedlings (one wound per soybean seedling, one mycelium cake per wound). Wrap the wounds with sterile cotton (wrap the mycelium cakes with the sterile cotton when wrapping the wounds).

[0068] 1.4 After wound inoculation, soybean potted plants were placed in a transparent humidity-controlled box and cultured under light for 12 hours. Culture conditions: temperature 25℃, light (3500K, 30000LUX) for 12 hours, relative humidity 80%. They were then placed in a greenhouse for 14 days. Greenhouse conditions: temperature 25℃, light (3500K, 30000LUX) for 16 hours, darkness for 8 hours, relative humidity 50%. Watering was done every 3 days, with approximately 200mL of sterile water per pot each time. The survival rate of soybean seedlings was then recorded.

[0069] The results are as follows Figure 5 As shown. From Figure 5It can be seen that, compared with the control group, the survival rate of soybean seedlings in the experimental group treated with Polyporus emblica PE-1 fermentation broth was significantly improved. This shows that Polyporus emblica PE-1 fermentation broth has a preventive or curative effect on soybean Phytophthora root rot.

[0070] 2. Experiment on the control effect of Polyporus emblica PE-1 fermentation broth on rice blast:

[0071] 2.1 Rice blast fungus ( Magnaporthe oryzae Preparation of Guy11 spore solution: Activated mycelial blocks (2mm × 2mm) of Guy11, the blast fungus of rice, were cut and inoculated onto SDC agar plates. Three plates were prepared and cultured at 28℃ under continuous light for 10 days. 3 mL of sterile water was added to each plate, and colonies were spread using a glass rod or brush. The mixture was filtered through a layer of Miracloth filter cloth, and the spores were collected in the same 50 mL tube. The spore concentration was adjusted to 100 spores / μL with sterile water.

[0072] 2.2 Fill cylindrical pots (13cm inner diameter and 13cm inner height) with 9cm of black soil, water with sterile water to moisten the black soil, and sow 30 germinated rice seeds (CCO39 variety, germinated to about 1.2mm in length) in each pot. Grow in a greenhouse at 25℃, with 12h light (3500K, 30000LUX fluorescent lamp) and 12h darkness, and 50% relative humidity for 10 days. Water once every 3 days, with about 200mL of sterile water per pot each time.

[0073] 2.3 In the experimental group (PE-1), 7 mL of *Polyporus emblica* PE-1 fermentation broth (8 mg / mL lyophilized *Polyporus emblica* PE-1 powder, prepared with sterile water) was evenly sprayed onto each rice seedling (on the leaves). The control group (CK) was sprayed with the same volume of sterile water. Both the experimental and control groups were replicated in 9 groups. After spraying, the seedlings were placed in a transparent humidity chamber and cultured under light for 12 hours. The culture conditions were: temperature 25℃, light (3500K, 30000LUX) for 12 hours, and relative humidity 80%.

[0074] 2.4 Spray each pot of rice seedlings evenly (on the leaves) with 7 mL of Guy11 spores (100 spores / μL) and place them in a greenhouse at 25℃, in darkness, and with a relative humidity of 70% for 24 hours. Then, grow them in a greenhouse at 25℃, with 12 hours of light (3500K, 30000LUX fluorescent lamp) followed by 12 hours of darkness, and a relative humidity of 90% for 6 days. Water them every 3 days, with about 200 mL of sterile water per pot each time. Then, calculate the area of ​​lesions on the rice seedlings (lesion area (%) = leaf lesion area of ​​1 pot of rice seedlings / total leaf area of ​​1 pot of rice seedlings * 100).

[0075] The results are as follows Figure 6 As shown. FromFigure 6 It can be seen that, compared with the control group, the number of rice seedling lesions in the experimental group treated with Polyporus emblica PE-1 fermentation broth was significantly reduced, indicating that Polyporus emblica PE-1 fermentation broth has a preventive or curative effect on rice blast.

[0076] 3. Field efficacy test of *Alternaria peltatum* PE-1 fermentation broth against wheat powdery mildew:

[0077] Test site and materials: Wheat field of Huai'an Academy of Agricultural Sciences, and the material variety was Huaimai 33.

[0078] Experimental Methods: Sowing was carried out on November 12, 2023, using machine row sowing at a seeding rate of 35 kg / mu. Field management was conducted according to conventional methods, without the application of pesticides. The experimental group (PE-1) was first sprayed with a bacterial preparation (prepared with sterile water, containing 8 mg / mL of freeze-dried *Polyporus emblica* PE-1 fermentation broth and 0.1 wt% surface dispersant Tween 80; sprayed on wheat ears and leaves; the same applies below) in late April of the following year (April 23, 2024), at a dosage of 1200 mL / 20 m². 2 A second application of the bacterial solution was performed 7 days later, at a dosage of 1200 mL / 20m². 2 The control group (CK) was sprayed with an equal volume of 0.1 wt% Tween 80 surface dispersant (prepared with sterile water). Both the experimental and control groups were configured with three replicates, each 20 m². 2 A survey of wheat powdery mildew severity was conducted in early to mid-May of the following year (May 20, 2024). Disease severity was assessed using the wheat powdery mildew grading standard, and the disease index and field control efficacy of *Polyporus emblica* PE-1 fermentation broth were calculated. Twenty plants were randomly selected from each group, and the disease severity of the first to third leaves from top to bottom on each plant were counted. Sixty leaves were counted from each group, and the disease index for each group was calculated.

[0079] Disease grading adopts the industry standard GB / T 17980.22-2000, "Guidelines for Field Efficacy Trials of Pesticides (I) - Grading Method for Powdery Mildew in Cereals Controlled by Fungicides (by Leaf):"

[0080] Level 0: No disease;

[0081] Grade 1: The area of ​​lesions accounts for less than 5% of the total leaf area;

[0082] Grade 3: The lesion area accounts for 6% to 15% of the total leaf area;

[0083] Level 5: The lesion area accounts for 16% to 25% of the total leaf area;

[0084] Level 7: The lesion area accounts for 26% to 50% of the total leaf area;

[0085] Level 9: The lesion area accounts for more than 50% of the total leaf area.

[0086] Formula for calculating the disease index:

[0087] Disease index = ∑(Number of diseased leaves at each level × Disease level) / (Total number of leaves surveyed × Highest disease level) × 100%

[0088] Formula for calculating prevention and control effect:

[0089] Prevention and control efficacy (%) = (Disease index of control group - Disease index of experimental group) / Disease index of control group × 100%

[0090] The results are shown in Tables 2 and 3. Figure 7 The results showed that the control effect of two applications of the bacterial solution on wheat powdery mildew was 70.0%.

[0091] Table 2

[0092]

[0093] Table 3

[0094]

[0095] 4. Field efficacy test of *Alternaria peltatum* PE-1 fermentation broth against wheat scab:

[0096] Test site and materials: Same as in Example 3, field efficacy test of fermentation broth of Polyporus emblica PE-1 against wheat powdery mildew (i.e., the same plot of land, statistical analysis of two diseases).

[0097] Experimental Methods: The field efficacy test of *Polyporus emblica* PE-1 fermentation broth against wheat powdery mildew was conducted as in Example 3. In early to mid-May of the following year (May 20, 2024), a wheat scab severity survey was conducted. Disease severity was statistically determined according to the wheat scab grading standard, and the disease index and field control effect of *Polyporus emblica* PE-1 fermentation broth were calculated. Six locations were surveyed for each group, and 100 wheat ears were collected from each location. The disease severity of 600 wheat ears from each group was counted, and the disease index for each group was calculated.

[0098] Disease grading adopts the grading method for Fusarium head blight (by ear) in the industry standard GB / T 15796-2011 Technical Specification for Monitoring and Forecasting of Wheat Fusarium Head Blight:

[0099] Level 0: No disease;

[0100] Grade 1: Diseased spikelets account for less than 25% of all spikelets;

[0101] Grade 2: Diseased spikelets account for 25%-50% of all spikelets;

[0102] Grade 3: Diseased spikelets account for 50%-75% of all spikelets;

[0103] Level 4: Diseased spikelets account for more than 75% of all spikelets.

[0104] Formula for calculating the disease index:

[0105] Disease index (%) = ∑(Number of diseased ears at each level × Disease level) / (Total number of ears surveyed × Highest disease level) × 100%

[0106] Formula for calculating prevention and control effect:

[0107] Prevention and control efficacy (%) = (Disease index of control group - Disease index of experimental group) / Disease index of control group × 100%

[0108] The results are shown in Tables 4 and 5. Figure 8 The results showed that the control effect of two applications of the bacterial solution on wheat scab was 51%.

[0109] Table 4

[0110]

[0111] Table 5

[0112]

[0113] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. A strain of *Alternaria emeraldica* PE-1, characterized in that, The fungus *Aspergillus violaceus* PE-1 is classified as *Aspergillus violaceus*. Clonostachys chloroleuca It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on February 2, 2024, with accession number CGMCC NO.41164.

2. The fungal agent prepared from *Alternaria peltatum* PE-1 according to claim 1, characterized in that, The bacterial agent is prepared by the following method: *Polyporus emblica* PE-1 is inoculated onto PDA medium plates and cultured at 25-28℃ in the dark for 12-14 days. The cultured *Polyporus emblica* PE-1 plates are cut into small pieces. 2-3 plates of 90mm diameter, 15mL PDA medium are added to every 1.5L MM liquid medium. The small pieces of *Polyporus emblica* PE-1 plates are placed in MM liquid medium and fermented at 25-28℃ in the dark at 160-180rpm for 5-7 days to obtain the fermentation broth. The fermentation broth is filtered through multi-layer gauze, and the filtrate is then filtered through a membrane to obtain the fermentation supernatant. The fermentation supernatant is freeze-dried to obtain *Polyporus emblica* PE-1 fermentation broth freeze-dried powder. When using, the bacterial agent is prepared with sterile water, and the bacterial agent contains 1-10 mg / mL of *Polyporus emblica* PE-1 fermentation broth freeze-dried powder.

3. The microbial agent according to claim 2, characterized in that, The filter membrane is a 0.45 μm filter membrane.

4. The application of *Aspergillus violaceus* PE-1 as described in claim 1 in the prevention of plant diseases, characterized in that... The plant diseases mentioned are soybean Phytophthora root rot, rice blast, wheat powdery mildew, and wheat scab.

5. The application of the microbial agent according to any one of claims 2-3 in the prevention of plant diseases, wherein the plant diseases are soybean Phytophthora root rot, rice blast, wheat powdery mildew, and wheat scab.

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