Polyspora emerald PE-1 and application thereof

Abstract

The invention discloses a strain of pyrethrum delavayi PE-1, the classification name of the pyrethrum delavayi PE-1 is pyrethrum delavayi, the pyrethrum delavayi PE-1 is pyrethrum delavayi, the pyrethrum delavayi is preserved in China General Microbiological Culture Collection Center (CGMCC), the preservation date is February 2, 2024, and the preservation number is CGMCC NO.41164. The pyrethrum delavayi has the advantages that the pyrethrum delavayi can be used for preparing the pyrethrum delavayi; the invention also discloses a microbial inoculum prepared from the strain. The invention further discloses application of the bacillus subtilis and a microbial agent prepared from the bacillus subtilis to prevention and treatment of plant diseases. The fermentation liquor of the screened euphorbia emerald PE-1 can widely induce the burst of reactive oxygen of plant immune markers in monocotyledonous plants and dicotyledonous plants, and can effectively prevent and treat various plant diseases such as phytophthora sojae root rot, rice blast, wheat powdery mildew, wheat scab and the like; new strain resources and technical support are provided 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] Currently, 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 large number of pathogenic fungi, the complex pathogenic population 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 pose a threat to 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. At present, 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 following technical solutions are adopted in the present application: In a first aspect, the present application provides a strain of Sporosarcina ureae PE-1, which is classified as Sporosarcina ureae. Clonostachys rosea f. catenulata It 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.

[0006] In a second aspect, the present application provides a microbial agent prepared from the above-mentioned Sporosarcina ureae PE-1.

[0007] Further, the bacterial agent is prepared by the following method: inoculating S. viridifaciens PE-1 to PDA culture medium plate, culturing at 25-28℃ in dark condition for 12-14d, cutting the S. viridifaciens PE-1 plate after culturing into small pieces, putting the small pieces of S. viridifaciens PE-1 plate into MM liquid medium, culturing at 25-28℃ in dark condition with 160-180rpm for 5-7d to obtain fermentation liquor; filtering the fermentation liquor with multi-layer gauze, and then filtering the obtained filtrate with filter membrane to obtain fermentation supernatant, freezing and drying the fermentation supernatant to obtain S. viridifaciens PE-1 fermentation liquor freeze-dried powder, and the bacterial agent is prepared by using sterile water, wherein the bacterial agent contains 1-10mg / mL of S. viridifaciens PE-1 fermentation liquor freeze-dried powder.

[0008] Further, the filter membrane is 0.45μm filter membrane.

[0009] The third aspect of the present application provides application of the above-mentioned S. viridifaciens PE-1 in preventing and treating plant diseases.

[0010] 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.

[0011] Further, the plant disease includes soybean Phytophthora root rot, rice blast, wheat powdery mildew and wheat scab.

[0012] The fourth aspect of the present application provides application of the above-mentioned bacterial agent in preventing and treating plant diseases.

[0013] 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.

[0014] Further, the plant disease includes soybean Phytophthora root rot, rice blast, wheat powdery mildew and wheat scab.

[0015] The present application has the following advantages: The application screens a strain of Gliocladium cymbiforme PE-1, and the fermentation liquor of the strain can widely induce the burst of plant immune markers active oxygen in monocotyledonous plants and dicotyledonous plants, and can effectively prevent and treat various plant diseases including soybean phytophthora root rot, rice blast, wheat powdery mildew and wheat scab, thereby providing new strain resources and technical support for the biological control of plant diseases.

[0016] Unlike traditional fungicides, the immune induction effect of the Gliocladium cymbiforme PE-1 on plants has a broad spectrum, and has a wide application prospect in various plant diseases. The Gliocladium cymbiforme PE-1 can significantly improve the disease resistance of plants and reduce the occurrence of diseases, including soybean phytophthora root rot, rice blast, wheat powdery mildew and wheat scab. The Gliocladium cymbiforme PE-1 has a remarkable effect in preventing and treating plant diseases, is environmentally friendly, avoids the persistent pollution of chemical pesticides to the environment, and has extremely high development and utilization value. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 It is a curve graph of the induction of the fermentation liquor of the strain PE-1 on the active oxygen burst of soybean yellowing seedling roots. RLU represents the fluorescence intensity.

[0018] Figure 2 It is a colony photograph of the strain PE-1.

[0019] Figure 3 It is a phylogenetic tree of the strain PE-1.

[0020] Figure 4 It is a curve graph of the induction of the fermentation liquor of the Gliocladium cymbiforme PE-1 on the active oxygen burst of different types of plants in Example 3. RLU represents the fluorescence intensity.

[0021] Figure 5 It is the prevention and treatment effect of the fermentation liquor of the Gliocladium cymbiforme PE-1 on soybean phytophthora root rot in Example 4.

[0022] Figure 6 It is the prevention and treatment effect of the fermentation liquor of the Gliocladium cymbiforme PE-1 on rice blast in Example 4.

[0023] Figure 7 It is the prevention and treatment effect of the fermentation liquor of the Gliocladium cymbiforme PE-1 on wheat powdery mildew in Example 4.

[0024] Figure 8 It is the prevention and treatment effect of the fermentation liquor of the Gliocladium cymbiforme PE-1 on wheat scab in Example 4. Biological material preservation information

[0025] The strain PE-1 is classified and named as Gliocladium cymbiforme Clonostachys rosea f. catenulata, and was preserved in China General Microbiological Culture Collection Center (CGMCC) at No. 3, Yuanmingyuan West Road, Beijing, China, on February 2, 2024, with a preservation number of CGMCC NO. 41164. DETAILED DESCRIPTION

[0026] The technical solutions of the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. The following embodiments facilitate better understanding of the present application, but do not limit the present application.

[0027] The test methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0028] The culture media involved in the following examples are as follows: MM liquid medium: glucose 10 g, sodium nitrate 6 g, potassium chloride 0.52 g, magnesium sulfate heptahydrate 0.312 g, potassium phosphate dibasic 1.52 g, vitamin B1 0.01 g, 1000X trace element solution 1 mL, deionized water to 1 L. Sterilize at 121 ℃ for 20 min.

[0029] 1000X trace element solution: magnesium sulfate heptahydrate 22 g, boric acid 11 g, manganese chloride tetrahydrate 5 g, cobalt chloride hexahydrate 1.7 g, copper sulfate pentahydrate 1 g, sodium manganate dihydrate 1.5 g, 50 g of EDTA tetrasodium, deionized water to 1 L. Store at 4 ℃.

[0030] PDA medium: peeled potatoes 200 g are cut into small pieces, 800 mL of deionized water is added to a pot, after the water boils, pour the potatoes into the pot, and cook for 15-20 min until the water is thick, add 20 g of glucose to a beaker, place two to three layers of gauze on the beaker, pour the thick potato liquid from the gauze into the beaker, and then add deionized water to 1 L, and add 20 g of agar powder. Sterilize at 121 ℃ for 20 min.

[0031] V8 medium: 1 g of CaCO3 is added to 100 mL of V8 juice, mixed and stirred on a magnetic stirrer for 10 min, centrifuged at room temperature at 3000 rpm for 5-7 min, the supernatant is taken, deionized water is added to 1 L, and 15 g of agar powder is added. Sterilize at 121 ℃ for 20 min.

[0032] SDC medium: 100 g (dry weight) of rice straw (about 1 cm long) is added with an appropriate amount of deionized water, boiled for 20 min, filtered with two layers of gauze, the residue is discarded, 40 g of corn flour is added to the filtrate, deionized water is added to 1 L, and 15 g of agar powder is added. Sterilize at 121 ℃ for 20 min.

[0033] The following examples relate to flat plates (petri dishes) having a diameter of 90 mm, unless otherwise specified.

[0034] Example 1 Isolation and identification of strain PE-1 1 Isolation of strain PE-1 1.1 Obtaining test material Healthy and well-grown soybean plants were selected from a soybean field in Lishui District, Nanjing City, Jiangsu Province, and root tissues were collected.

[0035] 1.2 Treatment of soybean root tissues A plurality of soybean root tissue blocks of about 2 mm x 2 mm were cut. The soybean root tissue blocks were sequentially soaked in 75 v / v% ethanol for 30 s, 2 m / v% sodium hypochlorite solution for 2 min, and then rinsed in sterile water for 3 times. The moisture on the soybean root tissue blocks was absorbed using sterile absorbent paper. 3-4 soybean root tissue blocks were placed on PDA medium plates containing 50 μg / mL rifampicin and 50 μg / mL ampicillin, and cultured at 25°C in the dark for 1-2 days. If colonies grew on the soybean root tissue blocks, they were immediately transferred to fresh PDA medium plates and cultured at 25°C in the dark.

[0036] 1.3 Activity screening The isolated strain (fungus, 5 mm fungus cake) was inoculated on a plate containing 15 mL of PDA medium and cultured at 25°C in the dark for 6-7 days. The cultured strain plate was cut into about 5 mm x 5 mm plate pieces (strain plate pieces) using a sterile scalpel and placed in MM liquid medium (2 plates of strain plate pieces were added to 1.5 L of MM liquid medium), and fermented in a shaker for 7 days under the conditions of 25°C, darkness, and 160 rpm. After fermentation, the strain fermentation broth was obtained, filtered with two layers of gauze, and the obtained filtrate was further filtered with a 0.45 μm filter membrane to obtain a strain fermentation supernatant. The strain fermentation supernatant was subjected to soybean yellow seedling root active oxygen detection: A cylindrical pot (32 cm in diameter and 35 cm in height) was filled with 20 cm of sterile vermiculite, and the vermiculite was moistened with sterile water. 50 soybeans (variety: He Feng 47) were sown in each pot, and the height of the vermiculite above the soybeans was about 1 cm. The soybeans were grown in a greenhouse at 25°C and a relative humidity of 70% under dark conditions for 3-4 days to obtain soybean yellow seedlings (the height of the soybean yellow seedlings was about 5 cm).

[0037] The 1 cm root tip of soybean etiolated seedling was cut off, and 8 cm soybean etiolated seedling roots without root tips were measured as test materials. The soybean etiolated seedling roots were cut into small pieces with a length of 5 mm with scissors, and 2 pieces were randomly selected and placed in a hole in a 96-well plate. Then 200 μL of sterile water was added to each hole. The 96-well plate was placed in an incubator at 25°C in the dark for 30 min. Then all the sterile water in each hole was sucked out, and 100 μL of active oxygen reaction solution was added as a treatment group (PE-1); 100 μL of MM liquid medium was added as a control group (CK). The active oxygen reaction solution (200 μL system) included horseradish peroxidase (P6782-10MG, SIGMA), ordinary type luminol (8511-5G, SIGMA), strain fermentation supernatant and sterile water; wherein the final concentration of horseradish peroxidase was 10 μg / mL (1000 times liquid mother liquor, prepared with sterile water), the final concentration of ordinary type luminol was 35.4 μg / mL (1000 times liquid mother liquor, prepared with DMSO), 100 μL of strain fermentation supernatant, and the rest was sterile water. After adding the active oxygen reaction solution, the 96-well plate was immediately placed in a fluorescence microplate reader, i.e. GLOMAX96 microplate luminometer (Promega, Madison, WI, USA), to detect the active oxygen burst of soybean etiolated seedling roots. Light was avoided during the test. Thus, strain PE-1 was screened, which can promote the active oxygen burst of soybean etiolated seedling roots and activate the immune response of soybean Figure 1 ).

[0038] 2 Identification of strain PE-1 2.1 Morphological identification Strain PE-1 was cultured on PDA medium at 25°C in the dark, as shown in Figure 2 , the colony was initially white, and the center of the mature colony gradually turned light green, and the edge remained white. The mycelium was in a cotton-like or villous shape, and the surface was moist or slightly dry (depending on the culture medium). When a large number of conidia were produced, the colony surface showed a powdery or granular structure (spores aggregated).

[0039] 2.2 Molecular biological identification 2.2.1 The genomic DNA of strain PE-1 was extracted by using the kit (DP320-50) of Tian Gen Biochemical Technology (Beijing) Co., Ltd.

[0040] 2.2.2 The EF1 -a, BenAThe DNA amplification reaction system was 25 μL, including 12.5 μL of 2x PCR Mix, 1 μL of forward and reverse primers (10 μM) respectively (the 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, a total of 34 cycles; finally 72 ℃ extension for 10 min.

[0041] 2.2.3 Sequencing results: the sequencing results of the strain PE-1 showed that the EF1 -a, BenA gene sequences are shown in SEQ ID NO. 1 and SEQ ID NO. 2 respectively.

[0042] 2.2.4 Comparing the obtained gene sequences on the NCBI database website, and constructing a phylogenetic tree Figure 3 ).

[0043] Therefore, it is inferred that the strain PE-1 is Gliocladium cymbiforme Clonostachys rosea f. catenulata ( C. rosea f. catenulata ), which has been preserved in the China General Microbiological Culture Collection Center (CGMCC) with the preservation number of CGMCC NO. 41164.

[0044] Table 1

[0045] Example 2 Preparation of Gliocladium cymbiforme PE-1 fermentation broth freeze-dried powder Activated fresh mycelium (5 mm mycelium cake) of Gliocladium cymbiforme PE-1 was picked and inoculated into a plate containing 15 mL of PDA medium, and then cultured at 25 ℃ in the dark for 14 d. The Gliocladium cymbiforme PE-1 plate after culture was cut into about 5 mm x 5 mm plate pieces (Gliocladium cymbiforme PE-1 plate pieces) with a sterile surgical knife, and then placed into MM liquid medium (2 plates of Gliocladium cymbiforme PE-1 plate pieces were added into 1.5 L of MM liquid medium), and then fermented and cultured at 25 ℃ in the dark at 160 rpm for 7 d to obtain a fermentation broth. The fermentation broth was filtered with two layers of gauze, and the obtained filtrate was further filtered with a 0.45 μm filter membrane to obtain a fermentation supernatant. The fermentation supernatant was freeze-dried (freeze-dried at -30 ℃, 3-5 Pa for 48 h) to obtain Gliocladium cymbiforme PE-1 fermentation broth freeze-dried powder.

[0046] Example 3 Broad-spectrum induction of plant immunity (burst of reactive oxygen species) of Gliocladium cymbiforme PE-1 fermentation broth in monocotyledonous plants and dicotyledonous plants Active oxygen burst is an important marker of plant immune induction. In this example, the fresh and intact leaves of monocotyledonous plants (wheat, corn, rice) and dicotyledonous plants (potato, tomato, soybean, Chinese cabbage, banana, cotton) were selected, and a puncher was used to punch a 2.5 mm diameter leaf disc on the front of the plant, or a knife was used to cut into a 2.5 mm x 2.5 mm square leaf, which was placed in a 96-well enzyme plate with 200 μL of sterile water in each well. The back of the leaf was facing up, and the entire 96-well enzyme plate was treated at room temperature overnight in the dark after the leaf disc was punched. On the 2nd day, all the sterile water in each well of the 96-well enzyme plate was gently aspirated with a sterile gun head, and the water was completely aspirated to avoid affecting the subsequent test results. A mixture containing ordinary lumi no, horseradish peroxidase, and Trichoderma atroviride PE-1 freeze-dried powder was prepared with sterile water. The final concentration of ordinary lumi no was 35.4 μg / mL (1000 times liquid, DMSO preparation), the final concentration of horseradish peroxidase was 10 μg / mL (1000 times liquid, sterile water preparation), and the final concentration of Trichoderma atroviride PE-1 freeze-dried powder was 8 mg / mL. The mixture was added to the above 96-well enzyme plate at 200 μL per well as the test group (PE-1). Then it was placed in a GLOMAX 96 microplate luminometer (Promega, Madison, WI, USA) to measure the luminescence value. The test process was carried out in the dark. The control group (CK) was set for each plant, i.e., the same volume of mixture containing ordinary lumi no (35.4 μg / mL) and horseradish peroxidase (10 μg / mL) was used instead of the mixture containing ordinary lumi no (35.4 μg / mL), horseradish peroxidase (10 μg / mL), and Trichoderma atroviride PE-1 freeze-dried powder (8 mg / mL). Each plant test group and control group had 12 replicates.

[0047] As Figure 4 The results showed that Trichoderma atroviride PE-1 fermentation broth can widely induce the burst of plant immune marker active oxygen in monocotyledonous plants and dicotyledonous plants, and has the potential to become a broad-spectrum elicitor for important crops such as Gramineae, Solanaceae, Leguminosae, Cruciferae, Musaceae, and Malvaceae.

[0048] Example 4: Prevention effect of Trichoderma atroviride PE-1 fermentation broth on plant diseases 1.1 The strong virulence strain of Phytophthora sojae was inoculated on the roots of soybean seedlings, and the roots were treated with Trichoderma atroviride PE-1 fermentation broth, and the control group was treated with sterile water. 1.1 The strong virulence strain of Phytophthora sojae was inoculated on the roots of soybean seedlings, and the roots were treated with Trichoderma atroviride PE-1 fermentation broth, and the control group was treated with sterile water. 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.

[0049] 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.

[0050] 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).

[0051] 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.

[0052] 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.

[0053] 2. Experiment on the control effect of Polyporus emblica PE-1 fermentation broth on rice blast: 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.

[0054] 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.

[0055] 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%.

[0056] 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).

[0057] The results are as follows Figure 6 As shown. From Figure 6It can be seen that compared with the control group, the test group applied with the fermentation broth of T. viride PE-1 has a significantly reduced disease spot area of rice seedlings, which shows that the fermentation broth of T. viride PE-1 has a preventive or curative effect on rice blast.

[0058] 3 Field efficacy test of T. viride PE-1 fermentation broth on wheat powdery mildew: Test site and material: wheat field of Huai'an Agricultural Academy, and the material variety is Huaimai 33.

[0059] Test method: sowing on November 12, 2023, machine drilling, sowing amount 35 kg / acre, field management according to the routine, and no pesticide is applied. The test group (PE-1) is sprayed with the broth preparation (sterile water preparation, containing 8 mg / mL T. viride PE-1 fermentation broth freeze-dried powder and 0.1wt% surface dispersant Tween 80) for the first time in late April of the next year (April 23, 2024), and the dosage is 1200 mL / 20 m 2 , and the second spraying is performed after 7 days, and the dosage is 1200 mL / 20 m 2 . The control group (CK) is sprayed with the same volume of 0.1wt% surface dispersant Tween 80 (sterile water preparation). The test group and the control group each have 3 repetitions, and each repetition has 20 m 2 . In early May of the next year (May 20, 2024), the severity of wheat powdery mildew is investigated, the disease grade is counted according to the wheat powdery mildew grading standard, and the disease index and the field control effect of T. viride PE-1 fermentation broth are calculated. Each group randomly counts 20 plants, and each plant counts the disease grade of the first to third leaves from top to bottom. Each group counts 60 leaves, and the disease index of each group is calculated.

[0060] The disease grading adopts the industry standard GB / T 17980.22-2000 Pesticide Field Efficacy Test Guidelines (I) Grading Method for Powdery Mildew Prevention and Cure of Bactericides (Leaf Unit): 0 grade: no disease; 1 grade: disease spot area accounts for less than 5% of the whole leaf area; 3 grade: disease spot area accounts for 6% to 15% of the whole leaf area; 5 grade: disease spot area accounts for 16% to 25% of the whole leaf area; 7 grade: disease spot area accounts for 26% to 50% of the whole leaf area; 9 grade: disease spot area accounts for more than 50% of the whole leaf area.

[0061] Disease index calculation formula: Disease index = ∑ (number of leaves of each grade x grade number) / (total number of leaves surveyed x highest grade number) x 100% The control effect calculation formula is: Control effect (%) = (disease index of the control group - disease index of the test group) / disease index of the control group x 100% The results are shown in Tables 2, 3 and Figure 7 The results show that the control effect of the wheat powdery mildew is 70.0% after two sprayings of the bacterial liquid preparation.

[0062] Table 2

[0063] Table 3

[0064] 4 Field efficacy test of Gliocladium virens PE-1 fermentation broth on wheat scab Test field and materials: same as the field efficacy test of Gliocladium virens PE-1 fermentation broth on wheat powdery mildew in Example 3 (i.e. the same field, statistics of two diseases).

[0065] Test method: same as the field efficacy test of Gliocladium virens PE-1 fermentation broth on wheat powdery mildew in Example 3. In the middle of May of the next year (May 20, 2024), the severity of wheat scab was investigated, and the disease grade was counted and the field control effect of Gliocladium virens PE-1 fermentation broth was calculated according to the wheat scab grading standard. 6 locations were investigated in each group, and 100 plant ears were taken from each location. The number of ears with disease in each group was counted, and the disease index of each group was calculated.

[0066] The disease grading adopts the grading method (in units of ears) of the industry standard GB / T 15796-2011 Wheat Scab Reporting Technical Specification: 0 level: no disease; 1 level: the number of diseased spikelets is less than 25% of the total spikelets; 2 level: the number of diseased spikelets is 25%-50% of the total spikelets; 3 level: the number of diseased spikelets is 50%-75% of the total spikelets; 4 level: the number of diseased spikelets is more than 75% of the total spikelets.

[0067] Disease index calculation formula: Disease index (%) = ∑ (number of diseased spikelets of each level x number of the disease level) / (total number of investigated ears x number of the highest disease level) x 100% Control effect calculation formula: Control effect (%) = (disease index of the control group - disease index of the test group) / disease index of the control group x 100% The results are shown in Tables 4, 5 and Figure 8The results show that the control effect of the two times of spraying the bacterial liquid preparation on the wheat scab is 51%.

[0068] Table 4

[0069] Table 5

[0070] The above detailed description of the specific embodiments of the present application is only as an example, and the present application is not limited to the above described specific embodiments. Any equivalent modifications and substitutions to the present application made by those skilled in the art are also within the scope of the present application. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present application should be covered within the scope of the present application.

Claims

1. A strain of Sporopolomyces turfosus PE-1, characterized in that, The taxonomic name of the Trichosporon virescens PE-1 is Trichosporon virescens Clonostachys rosea f. catenulata , which is preserved in the China General Microbiological Culture Collection Center on February 2, 2024, and the preservation number is CGMCC NO. 41164.

2. The bacterial agent prepared from Clonostachys rosea f. catenulata PE-1 according to claim 1.

3. The bacterial agent of claim 2, wherein The bacterial agent is prepared by the following method: Clonostachys rosea f. catenulata PE-1 is inoculated on PDA medium plate, and cultured at 25-28℃ in dark for 12-14 days; the cultured Clonostachys rosea f. catenulata PE-1 plate is cut into small pieces, and 2-3 pieces of 90mm in diameter are put into 1.5L MM liquid medium, and fermented at 25-28℃ in dark at 160-180rpm for 5-7 days to obtain fermentation broth; the fermentation broth is filtered with multi-layer gauze, and the obtained filtrate is further filtered with filter membrane to obtain fermentation supernatant; the fermentation supernatant is freeze-dried to obtain Clonostachys rosea f. catenulata PE-1 fermentation broth freeze-dried powder; when used, the bacterial agent is prepared by using sterile water to dissolve the Clonostachys rosea f. catenulata PE-1 fermentation broth freeze-dried powder to a concentration of 1-10mg / mL.

4. The bacterial agent of claim 3, characterized in that, The filter membrane is a 0.45μm filter membrane.

5. The use of Clonostachys rosea f. catenulata PE-1 in the prevention and treatment of plant diseases according to claim 1.

6. Use according to claim 5, characterized in that, The plants include monocotyledonous plants and dicotyledonous plants; the monocotyledonous plants include Poaceae, and the dicotyledonous plants include Solanaceae, Leguminosae, Cruciferae, Musaceae and Malvaceae; the Poaceae include wheat, corn and rice, the Solanaceae include potato and tomato, the Leguminosae include soybean, the Cruciferae include Chinese cabbage, the Musaceae include banana, and the Malvaceae include cotton.

7. Use according to claim 5, characterized in that, The plant diseases include soybean Phytophthora root rot, rice blast, wheat powdery mildew and wheat scab.

8. The use of the bacterial agent according to any one of claims 2-4 in the prevention and treatment of plant diseases.

9. Use according to claim 8, characterized in that, The plants include monocotyledonous plants and dicotyledonous plants; the monocotyledonous plants include Poaceae, and the dicotyledonous plants include Solanaceae, Leguminosae, Cruciferae, Musaceae and Malvaceae; the Poaceae include wheat, corn and rice, the Solanaceae include potato and tomato, the Leguminosae include soybean, the Cruciferae include Chinese cabbage, the Musaceae include banana, and the Malvaceae include cotton.

10. Use according to claim 8, characterized in that, The plant diseases include soybean Phytophthora root rot, rice blast, wheat powdery mildew and wheat scab.

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