Application of penicillium roqueforti pr1 secondary metabolites and effective components in prevention and treatment of plant bacterial diseases

Abstract

The application discloses application of a secondary metabolite of Penicillium roqueforti PR1 and an effective component thereof in prevention and treatment of plant bacterial diseases, and belongs to the technical field of bactericides. The secondary metabolite of Penicillium roqueforti PR1 (Z44-K1) prepared by the application and ethyl-alpha-D-glucoside (Z44-K11) and ethyl-beta-D-glucopyranoside (Z44-K12) contained in the secondary metabolite have good prevention and control effects on rice bacterial leaf blight bacteria. The indoor prevention effect of Z44-K1 reaches 44.29% at a concentration of 50 ng / mL; the indoor prevention effect of Z44-K11 on rice bacterial leaf blight reaches 60.87% at 50 ng / mL; and the indoor prevention effect of Z44-K12 reaches 59.88% at 10 ng / mL. As plant immune inducers, the secondary metabolite and the effective component thereof are extremely low in dosage, safe, green and efficient, and provide a new idea for prevention and treatment of plant bacterial diseases.

Description

Technical Field

[0001] This invention relates to the field of fungicide technology, specifically to the application of secondary metabolites of Penicillium erythropoiesis-producing fungus PR1 and its active ingredients in the prevention and control of bacterial plant diseases. Background Technology

[0002] Plant immune inducers activate the plant's own immune system, enabling the plant to potentially control diseases. These agents typically lack direct bactericidal activity but induce disease resistance, thereby increasing the plant's inherent disease resistance, reducing the use of chemical pesticides, and contributing to maintaining agricultural ecological balance. Currently discovered plant immune inducers include amino oligosaccharides, chitosan oligosaccharides, and protein-based substances, but their application remains relatively limited and warrants further research. Among these, discovering novel plant immune activators from natural products to control bacterial plant diseases is of significant research importance. Bacterial plant diseases are common and damaging in agricultural production, infecting various food and cash crops such as tobacco, tomatoes, peppers, cucumbers, rice, and potatoes. They often lead to stunted growth, leaf necrosis or wilting, reduced fruit quality, and yield losses. Existing public information generally considers bacterial plant diseases as difficult to control, noting that various bacterial pathogens can cause substantial agricultural economic losses. Because plant bacterial diseases are characterized by rapid spread, wide prevalence, significant susceptibility to environmental conditions, and high difficulty in prevention and control, developing safe, green, and effective plant disease control technologies has always been an important research direction in the field of plant protection.

[0003] Rice, China's largest and the world's third-largest food crop, is susceptible to bacterial blight, a common bacterial disease that causes losses ranging from 10% to 30%, and in severe cases, up to 50% or even over 90%. This disease is caused by the fungus *Xanthomonas oryzae* pv. oryzae, and typically begins at the leaf tip or margin, initially appearing as small, translucent yellow spots that later develop into wavy, yellowish-green or grayish-green lesions. While chemical control has played a crucial role in controlling the occurrence and spread of bacterial blight, it also faces challenges such as pathogen resistance, pesticide residues, and environmental pollution. Therefore, researching and developing novel, naturally derived, green biological pesticides, such as plant immune inducers, is of great significance.

[0004] Currently, the control of bacterial plant diseases still mainly relies on a comprehensive approach including chemical agents, resistant varieties, and cultivation management. However, existing chemical control methods generally suffer from limited efficacy, susceptibility to application timing and environmental conditions, and the potential for pesticide residues and environmental stress with long-term use. Meanwhile, the discovery of novel plant protectants from microbial, especially fungal, active substances has become an important direction for the development of biopesticides and plant immune regulators. Publicly available patents indicate that active products derived from Penicillium fungi can be used for plant disease control. For example, active products from Penicillium fungi can act as plant immune inducers, enhancing plant resistance to diseases. *Penicillium erythropoiesis-1* strain and its mycelial extracts can be used to promote crop growth or increase yield, improve tomato resistance to root-knot nematodes and improve fruit quality, and can also act as plant immune inducers to enhance plant resistance to bacterial diseases. However, existing public applications of *Penicillium erythropoiesis-1* mainly focus on promoting growth and yield, improving fruit quality, and enhancing resistance to root-knot nematodes; research on its application in the control of bacterial plant diseases remains limited. The control of bacterial plant diseases places high demands on the safety, environmental friendliness, application cost, and ability to induce plant systemic resistance of active substances. Therefore, it is necessary to develop a secondary metabolite of Penicillium erythropoiesis-producing strain PR1 with a clear source, feasible preparation, and good control effect on bacterial plant diseases, and to develop its application, in order to solve the technical problems of limited effectiveness, insufficient greenness, and poor sustainable application of existing bacterial plant disease control methods. Summary of the Invention

[0005] The purpose of this invention is to address the aforementioned limitations of existing technologies by providing the application of secondary metabolites of *Penicillium erythropoiesis* PR1 and their active ingredients in the control of bacterial plant diseases. This invention, through research, has discovered the control efficacy of *Penicillium erythropoiesis* PR1 secondary metabolites and their terminal isomers, ethyl-α-D-glucoside and ethyl-β-D-glucopyranoside, against rice bacterial blight pathogens. As plant immune inducers, they are not only used in extremely low doses but are also safe, green, and highly effective, providing a new approach to the control of bacterial plant diseases.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the application of secondary metabolites of Penicillium erythropoiesis-producing fungus PR1 in the control of bacterial plant diseases, wherein the secondary metabolites of Penicillium erythropoiesis-producing fungus PR1 are prepared by the following method: (1) The ethanol extract of Penicillium PR1 mycelium was concentrated to a viscous state and then freeze-dried to obtain a crude extract. (2) The crude extract was successively extracted with petroleum ether, CH2Cl2 and ethyl acetate, and the ethyl acetate extract was concentrated to obtain the extract solution; (3) Add anhydrous ethanol to the extract to make the ethanol content in the solution reach 50%. After alcohol extraction, let stand and centrifuge to separate the first supernatant. Add anhydrous ethanol to the first supernatant to make the ethanol content in the solution reach 75%. After alcohol extraction, let stand and centrifuge to separate the second supernatant. After the second supernatant is concentrated and dried, it is extracted with methanol. After standing and centrifuging, the third supernatant is separated. The third supernatant is concentrated to a viscous state and freeze-dried to obtain the alcohol extract. (4) The alcohol extract was prepared into a solution with methanol and separated and purified by semi-preparative HPLC. The component with the second highest content was selected and passed through a C18 column to obtain the secondary metabolites of Penicillium erythropoiesis.

[0007] Preferably, in step (1), the ethanol extract of the Penicillium pr1 mycelium is prepared by the following method: The *Penicillium erythropoiesis-producing* PR1 was sequentially cultured and activated, and then seed cultured to obtain a seed liquid. The seed liquid was inoculated into a fermentation medium for fermentation to obtain a fermentation broth. The fermentation broth was centrifuged and filtered to obtain mycelium. Finally, the mycelium was dried, pulverized, and then extracted with ethanol by cold soaking followed by ultrasonic extraction to obtain an ethanol extract of the *Penicillium erythropoiesis-producing* PR1 mycelium.

[0008] Preferably, the erythropoietic Penicillium PR1 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 13189.

[0009] Preferably, in step (1), the concentration is to add the ethanol extract of Penicillium pr1 mycelium to a rotary evaporator, set the water bath temperature to 50°C, the vacuum degree to 20 mbar, and the ethanol condensation temperature to -8°C; the freeze drying is to pre-freeze in a -80°C refrigerator for 12 h, take it out, punch holes, and freeze dry in a freeze dryer for 72 h.

[0010] Preferably, in step (3), the settling temperature is 4°C and the settling time is 5h; the centrifugation speed is 5000 r / min and the settling time is 3min.

[0011] Preferably, in step (4), the concentration of methanol is 50 wt%; and the concentration of the solution is 0.84 g / mL.

[0012] Preferably, the concentration of the secondary metabolite of Penicillium erythropoiesis-producing strain PR1 is 50 ng / mL.

[0013] Preferably, the plant is rice, and the bacterial disease is bacterial blight caused by Xanthomonas.

[0014] In a second aspect, the present invention provides the application of an active ingredient of a secondary metabolite of Penicillium rubrum PR1 in the prevention and control of bacterial diseases of plants, wherein the active ingredient is ethyl-α-D-glucoside or ethyl-β-D-glucopyranoside.

[0015] Preferably, the concentration of the ethyl-α-D-glucoside used is 50 ng / mL; the concentration of the ethyl-β-D-glucopyranoside used is 10 ng / mL; the plant is rice; and the bacterial disease is bacterial blight caused by Xanthomonas.

[0016] The beneficial effects of this invention are: (1) This invention discovers, through research, that *Penicillium rubrum* is produced. Penicillium rubens Secondary metabolites of PR1 have the effect of resisting rice bacterial blight. The control effect of PR1 on rice bacterial blight was tested by leaf cutting inoculation and spraying of pesticides. The activity of enzymes related to the defense response of rice leaves and the regulatory effect of salicylic acid were also measured.

[0017] (2) This invention produces Penicillium erythropoiesis Penicillium rubens Analysis of the secondary metabolites of PR1 revealed the control effects of the terminal isomers ethyl-α-D-glucoside and ethyl-β-D-glucopyranoside on rice bacterial blight pathogens. Furthermore, ethyl-β-D-glucopyranoside showed better efficacy than ethyl-α-D-glucoside, providing a scientific basis for the prevention and treatment of plant bacterial diseases.

[0018] (3) The Z44-K1 prepared in this invention has a good control effect on rice bacterial blight caused by Xanthomonas strain PXO99A, with an indoor control efficacy of 44.29% at a concentration of 50 ng / mL. The main component Z44-K11 (ethyl α-D-glucoside) contained in Z44-K1 has an indoor control efficacy of 60.87% against rice bacterial blight at 50 ng / mL; Z44-K12 (ethyl β-D-glucopyranoside) has an indoor control efficacy of 59.88% at 10 ng / mL.

[0019] (4) The results of the detection of the regulatory effects of Z44-K1, Z44-K11 and Z44-K12 prepared in this invention on the activity of enzymes related to the defense response of rice leaves and the salicylic acid showed that K1-50 significantly upregulated the POD activity level in rice; K11-50 significantly upregulated the PPO and POD activity levels in rice; K12-10 significantly upregulated the CAT activity level and salicylic acid content in rice. K1, K11 and K12 improved the disease resistance of rice and resisted the infection of Xanthomonas by inducing the expression of plant defense-related enzymes and salicylic acid signaling pathway. Attached Figure Description

[0020] Figure 1Z44-K1 1 H-NMR spectra (chemical shift 1-5 ppm); Figure 2 Z44-K1 13 C-NMR spectra (chemical shift 0-120 ppm); Figure 3 HMBC spectrum of Z44-K1; Figure 4 HSQC spectrum of Z44-K1; Figure 5 COSY spectrum of Z44-K1; Figure 6 NOE spectrum of Z44-K1; Figure 7 The control effect of different concentrations of Penicillium erythropoiesis-producing PR1 secondary metabolites; Figure 8 Photos of different concentrations of Penicillium erythropoiesis-producing PR1 used to control bacterial leaf blight in rice; Figure 9 The control effects of Z44-K11 and Z44-K12 at different concentrations; Figure 10 Photos of Z44-K11 and Z44-K12 at different concentrations for controlling bacterial blight in rice; Figure 11 The statistical analysis of the enzyme activities and salicylic acid content of different agents on the defense response of rice leaves, including (a) PPO activity; (b) SOD activity; (c) POD activity; (d) CAT activity; and (e) SA content. Detailed Implementation

[0021] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0022] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0023] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0024] Example 1: (1) Preparation of ethanol extract of Penicillium pruriens mycelium PR1: Take 200 g of peeled potato, cut it into strips, and boil it in 1000 mL of water for 20-30 min. While hot, remove the coarse fiber from the potato with 2-3 layers of gauze. Add 20 g each of agar and glucose, and stir evenly with a glass rod to prepare PDA medium. Sterilize at 120℃ for 30 min and set aside for use.

[0025] The target strain PR1, preserved at 4℃, was inoculated onto prepared PDA medium and cultured at 25℃ for 6 days. Then, it was inoculated into an Erlenmeyer flask containing 50 mL of seed culture medium (PDA liquid medium) and cultured on a rotating shaker at 25℃ and 180 r / min for 3 days as seed culture. 200 g of peeled potatoes were chopped and boiled in 1000 mL of distilled water for 30 min. The potato pieces were filtered out, and 1000 mL of the potato extract was used to dissolve 1 g of yeast extract, 3 g of peptone, 15 g of glucose, and 17 g of agar. Sufficient fermentation medium was prepared and sterilized at 120℃ for 30 min before use.

[0026] Inoculate 10% of the culture medium into 150 mL of fermentation medium, culture under the same conditions for 5 days, then terminate the fermentation to obtain the final fermentation broth.

[0027] The ethanol extraction procedure was as follows: the fermentation broth was centrifuged and filtered to obtain mycelium. The mycelium was dried at a constant temperature of 60°C to remove excess moisture. The dried mycelium was then pulverized to facilitate subsequent extraction. An appropriate amount of ethanol was added at a ratio of 20% of the mycelium weight, and the mixture was cold-soaked for 24 hours, followed by ultrasonic extraction for 1 hour. This extraction was repeated three times, and the filtrates were combined. The amount of ethanol added was 20% of the mycelium weight; the amount of ethanol added for each extraction was the same as the first time to ensure consistency of extraction conditions.

[0028] (2) Preparation of crude extract: The PR1 extract was transferred to a rotary evaporator, the water bath temperature was set to 50℃, the vacuum to 20mbar, and the ethanol condensation temperature to -8℃. The sample was concentrated to a viscous state, covered with a sealing film, and pre-frozen in a -80℃ freezer for 12 h. After removal, holes were punched, and the sample was freeze-dried in a freeze dryer for 72 h until the sample was completely dry to obtain crude extract Z4. The obtained samples were weighed and recorded, and then sealed and stored in a desiccator.

[0029] (3) Preparation of extract: 71 g of crude extract Z4 was added to 450 mL of deionized water and ultrasonically cleaned for 15 min at an ultrasonic frequency of 40 kHz and a temperature of 30 °C to assist dissolution, resulting in a solution with a concentration of 0.158 g / mL. 450 mL of petroleum ether was added at a volume ratio of 1:1 (water:petroleum ether) for extraction, followed by vacuum concentration. 450 mL of dichloromethane was added at a volume ratio of 1:1 (water:dichloromethane) for extraction again, followed by vacuum concentration. 450 mL of ethyl acetate was added at a volume ratio of 1:1 (water:ethyl acetate) for extraction again, followed by vacuum concentration.

[0030] (4) Preparation of alcohol extract: Add 500 mL of anhydrous ethanol to the extract to make the ethanol content in the solution reach 50% vt%, and sonicate for 15 min at an ultrasonic frequency of 40 KHz and a temperature of 30℃ to make it fully dissolved. After standing in a 4℃ refrigerator for 5 h, the solution was centrifuged at 5000 r / min for 3 min to obtain the first supernatant. The precipitate was freeze-dried to obtain crude extract Z41. 1000 mL of anhydrous ethanol was added to the first supernatant to make the ethanol content of the solution reach 75 vt%, and the solution was sonicated for 15 min under the same conditions. After standing in a 4℃ refrigerator for 5 h, the solution was centrifuged at 5000 r / min for 3 min to obtain the second supernatant. The precipitate was freeze-dried to obtain crude extract Z42. The second supernatant was concentrated and dried to obtain 51 g of crude extract, which was transferred to 250 mL of methanol and dissolved under the same sonic conditions. After standing in a 4℃ refrigerator for 5 h, the solution was centrifuged under the same conditions to obtain the third supernatant. The precipitate was dried to obtain crude extract Z43. The third supernatant was concentrated to a viscous state by rotary evaporation at a temperature of 35-40℃ and a rotation speed of 30-70 rpm, and then freeze-dried to obtain crude extract Z44. Crude extracts Z41, Z42, Z43, and Z44 were ground into powder, weighed, recorded, and stored in a desiccator after sealing. Sample Z44 yielded 42.532 g, with an extraction rate of 59.90%.

[0031] (5) Preparation of secondary metabolites of Penicillium erythropoiesis-producing strain PR1: Z44 was prepared into a solution with a concentration of 0.84 g / mL using methanol-water (volume ratio 1:1). Z44 was then separated and purified using a semi-preparative HPLC system (Waters 2996) to obtain 14 active metabolites and other components (Z44-QT). Z44-6 had the highest content, followed by peaks Z44-4, Z44-13, and Z44-14.

[0032] The Z44-4 fraction (218.0 mg) was prepared by a methanol-water gradient (methanol and water volume ratio of 1:9) and a C18 column to obtain fraction Z44-K1 (tR=3.6min, 82.8mg), which is a secondary metabolite of Penicillium erythropoiesis, hereinafter referred to as K1.

[0033] (6) Preparation of the effective components of secondary metabolites of Penicillium erythropoiesis-producing fungus PR1: from Figures 1-6 As can be seen, analysis of K1 obtained in Example 1 showed no obvious other impurity peaks. NMR analysis indicated that K1 was a mixed sample of two compounds, mainly composed of ethyl-α-D-glucoside and ethyl-β-D-glucopyranoside. K1 was separated into Z44-K1-1 (a mixture of ethyl-α-D-glucoside Z44-K11 and ethyl-β-D-glucopyranoside Z44-K12) by methanol-water (methanol to water volume ratio 1:18) 5PFP column chromatography. t R Two components (5.0 min, 29.0 mg).

[0034] Z44-K11 (Ethyl-α-D-glucoside, molecular formula C8H) 16 The structural formula of O6 (molecular weight 208) is: .

[0035] Z44-K12 (Ethyl-β-D-glucoside, molecular formula C8H) 16 The structural formula of O6 (molecular weight 208) is: .

[0036] right Figures 1-6 The NMR data of the two compounds obtained from the NMR spectrum analysis are shown in Table 1.

[0037] Table 1. Analysis of NMR data As can be seen from Table 1, the main difference between the two is that the terminal hydrogen of ethyl-α-D-glucoside is an axial bond with a small coupling constant; while the terminal hydrogen of ethyl-β-D-glucopyranoside is an equatorial bond with a large coupling constant. Figure 1 NMR data analysis indicates that K1 is a mixture of ethyl-α-D-glucoside and ethyl-β-D-glucopyranoside, and the integral of the proton NMR spectrum shows that the ratio of the two is approximately 1:0.44.

[0038] Since ethyl-α-D-glucoside Z44-K11 and ethyl-β-D-glucopyranoside Z44-K12 are difficult to separate, the test was conducted using ethyl-α-D-glucoside standard (CAS: 19467-01-7, hereinafter referred to as K11) and ethyl-β-D-glucopyranoside standard (CAS: 3198-49-0, hereinafter referred to as K12).

[0039] Experiment Example 1: Test on the control effect of rice bacterial blight Inoculation with Xanthomonas oryzae PXO99, a pathogen causing white leaf blight, was performed by cutting leaves. A (Gong Liping. Xanthomonas orbacterium bacterial blight strain PXO99 A A Preliminary Study on the Mechanism of Toxicity Differences in 146 Rice Varieties [D]. Shandong Agricultural University, 2023.) The effect of K1 on rice resistance to bacterial blight was analyzed by applying different concentrations of pesticide to rice leaves. The specific experimental procedure was as follows: (1) Plant cultivation: Conventional rice (Mizuni Nipponbare) cultivation was carried out in a climate chamber at 28 ℃ and 50%-60% humidity; (2) Inoculate Xanthomonas into liquid NB medium and incubate at 28 ℃ for 2-3 days; (3) Centrifuge the cultured bacterial solution at 4000 rpm for 5 min and discard the supernatant; wash the bacterial cells once with 10 mM MgCl2, and then resuspend the bacterial solution again with MgCl2 solution; (4) Adjusting OD 600 Value: Adjust the OD600 value of the bacterial cells to 0.5 using sterile water; (5) Select plant leaves: Select disease-free and insect-free 14-day-old rice seedlings with the same growth and inoculate the leaves at the same position.

[0040] (6) Leaf cutting inoculation: Disinfect scissors, dip them in bacterial solution, and cut the leaves 3 cm from the leaf tip. Ensure that the inoculation position is uniform.

[0041] (7) Keep the leaves moist: spray with an appropriate amount of sterile water.

[0042] As shown in Table 2, this experiment included 7 treatments, with 24 rice plants inoculated for each treatment. The pesticide was prepared according to the required concentration. The pesticide was sprayed first (10 mL / plant), and 4 hours later, leaves were cut for inoculation. After 14 days, the length of lesions on the inoculated leaves was counted and compared, and the control effect of each treatment group was calculated as ((1 - average lesion length of treatment group / average lesion length of control group) × 100%). The results are shown in Table 3 and... Figure 7 , Figure 8 .

[0043] Table 2 Test Treatments and Drug Dosage Table 3. Length of lesions and indoor preventive effect of rice 14 days after inoculation. According to Table 3 and Figures 7-8 It can be seen that the optimal concentration of K1 for control is 50 ng / mL, with a control efficiency of 44.29%. When the K1 concentration increases to 100 ng / mL: the dose of the inducer exceeds the plant's immune adaptation threshold, resulting in immune receptor desensitization and signaling pathway inactivation; at the same time, the continuous excessive immune initiation consumes a large amount of plant growth and stress-resistance metabolic energy, induces abnormal accumulation of reactive oxygen species (ROS), produces an immune autoinhibition effect, and the expression level of disease resistance-related genes drops significantly. Finally, the lesion length is close to that of the blank control. When the K1 concentration increases to extremely high concentrations of 500-1000 ng / mL, the plant initiates basic stress compensation, and the control efficacy recovers slightly, but it is still far lower than the optimal low concentration treatment, which is a typical bell-shaped dose effect.

[0044] Experimental Example 2: Test on the control effect of extracts of secondary metabolites of Penicillium rubrum PR1 on rice bacterial blight As shown in Table 4, this experiment consisted of 12 treatments. The experimental method was the same as in Experiment 1. The control effects of each treatment are shown in Table 5. Figures 9-10 .

[0045] Table 4. Dosage of Pharmaceuticals Table 5. Lesion length and indoor prevention effect As shown in Table 5 and Figures 9-10 As shown, the optimal concentration of K11 for control was 50 ng / mL, with a control efficiency of 60.87%; the optimal concentration of K12 for control was 10 ng / mL, with a control efficiency of 59.88%. Since the lesion length in the K12 treatment group (50 ng / mL) was greater than that in the blank control, it is listed in Table 4. Figure 8 Its prevention and control effect was marked as 0. K12 and reagent K1 in Experiment 1 have a typical bell-shaped dose effect.

[0046] Experimental Example 3: The regulatory effect of the agent on the activity of enzymes related to the defense response in rice leaves and salicylic acid. (1) Experimental procedure: K1 (50 ng / mL), K11 (50 ng / mL), and K12 (10 ng / mL) prepared in Example 1 were sprayed according to the method of Example 1, and water was sprayed as a blank control. They were recorded as K1-50, K11-50, K12-10 and CK groups, respectively. Four hours after spraying, the leaves were cut and inoculated with Xanthomonas oryzae bacterial blight pathogen (Xanthomonas oryzae) PXO99. AApply to rice leaves after spraying with different concentrations of pesticide.

[0047] 48 hours after spraying and inoculation, rice leaves were collected for enzyme activity testing. Polyphenol oxidase (PPO) activity: The activity was expressed as the change in absorbance per unit time at 410 nm using the catechol method. PPO catalyzes the oxidation of catechol to generate colored quinone products.

[0048] Superoxide dismutase (SOD) activity: The WST-8 method was used. SOD inhibits the formation of chromogenic products by scavenging superoxide anion free radicals. The enzyme activity was calculated by the inhibition rate of absorbance at 450 nm.

[0049] Peroxidase (POD) activity: The guaiacol method was used. POD catalyzes the oxidation of guaiacol by H2O2 to produce a colored product. The enzyme activity is expressed as the change in absorbance per unit time at 470 nm.

[0050] Catalase (CAT) activity: The ammonium molybdate colorimetric method was used. CAT decomposes H2O2, and the remaining H2O2 forms a yellow complex with ammonium molybdate. The enzyme activity was calculated by the change in absorbance at 405 nm.

[0051] Salicylic acid (SA) content: The salicylic acid content was calculated based on absorbance values ​​using enzyme-linked immunosorbent assay (ELISA) based on a competitive immune reaction. The results are shown below. Figure 11 .

[0052] (2) Results analysis: 1) Polyphenol oxidase (PPO) activity Compared with the control group (CK), the PPO activities of K1-50 and K12-10 were not significantly different from those of CK; however, the PPO activity of K11-50 was significantly increased, indicating that K11-50 can significantly upregulate the activity level of polyphenol oxidase (PPO) in rice. The increase in PPO can synthesize a large amount of quinones to inhibit microbial infection, and can bind to proteases secreted by pathogens to inhibit their pathogenicity and enhance plant disease resistance.

[0053] 2) Superoxide dismutase (SOD) activity Superoxide dismutase (SOD) is the first line of defense in a plant's antioxidant system, and its activity directly reflects the plant's ability to scavenge reactive oxygen species (ROS). When pathogens infect plants, they produce large amounts of ROS as a defense signal through an "oxidative burst." SOD participates in regulating ROS concentration to prevent excessive oxidative damage. Experiments showed that compared to the control (CK), no increase in SOD activity was observed in any of the three reagents, and some treatments showed lower SOD activity than the CK.

[0054] 3) Peroxidase (POD) activity Compared with the control group (CK), the POD activity of K1-50 and K12-10 was significantly increased; K11-50 had the highest POD activity, indicating that K11-50 can significantly upregulate the activity level of peroxidase (POD) in rice. The increase of POD accelerates the catalytic products, which can directly inhibit the growth of pathogens. At the same time, it participates in the cell wall lignification process, increases lignin content, forms a physical barrier, and enhances the plant's disease resistance.

[0055] 4) Catalase (CAT) activity Compared with the control group, the CAT activity of K12-10 was significantly increased in all treatment groups, indicating that K12-10 can significantly upregulate the activity level of catalase (CAT) in rice. The increase of CAT accelerates the removal of H2O2 in leaves, delays membrane lipid peroxidation, prolongs the functional period of leaves, and reduces pathogen infection damage.

[0056] 5) Salicylic acid (SA) content Compared with the control group, the salicylic acid content of K11-50 and K12-10 was significantly increased in each treatment group, indicating that K11-50 and K12-10 can significantly upregulate the salicylic acid content in rice. The increase in SA activates the expression of pathogenesis-related proteins (PR proteins), such as PR-1 and PR-2 (β-1,3-glucanase), thereby enhancing the plant's resistance to pathogens.

[0057] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of secondary metabolites of Penicillium erythropoiesisrum PR1 in the control of bacterial plant diseases, characterized in that, The secondary metabolites of the PR1 erythropoietin fungus were prepared by the following method: (1) The ethanol extract of Penicillium PR1 mycelium was concentrated to a viscous state and then freeze-dried to obtain a crude extract. (2) The crude extract was successively extracted with petroleum ether, CH2Cl2 and ethyl acetate, and the ethyl acetate extract was concentrated to obtain the extract solution; (3) Add anhydrous ethanol to the extract to make the ethanol content in the solution reach 50%. After alcohol extraction, let stand and centrifuge to separate the first supernatant. Add anhydrous ethanol to the first supernatant to make the ethanol content in the solution reach 75%. After alcohol extraction, let stand and centrifuge to separate the second supernatant. After the second supernatant is concentrated and dried, it is extracted with methanol. After standing and centrifuging, the third supernatant is separated. The third supernatant is concentrated to a viscous state and freeze-dried to obtain the alcohol extract. (4) The alcohol extract was prepared into a solution with methanol and separated and purified by semi-preparative HPLC. The component with the second highest content was selected and passed through a C18 column to obtain the secondary metabolites of Penicillium erythropoiesis.

2. The application according to claim 1, characterized in that, In step (1), the ethanol extract of the Penicillium PR1 mycelium is prepared by the following method: The Penicillium erythropoiesis strain PR1 was successively activated by culture and then cultured in seed culture to obtain seed liquid; The seed culture was inoculated into a fermentation medium for fermentation to obtain a fermentation broth; the fermentation broth was centrifuged and filtered to obtain mycelium; finally, the mycelium was dried, pulverized, and then extracted with ethanol by ultrasonic extraction to obtain an ethanol extract of the Penicillium PR1 mycelium.

3. The application according to claim 2, characterized in that, The erythropoietic Penicillium PR1 strain has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO. 13189.

4. The application according to claim 1, characterized in that, In step (1), the concentration involves adding the ethanol extract of Penicillium pr1 mycelium to a rotary evaporator, setting the water bath temperature to 50°C, the vacuum degree to 20 mbar, and the ethanol condensation temperature to -8°C; the freeze-drying involves pre-freezing in a -80°C refrigerator for 12 h, taking it out, punching holes in it, and freeze-drying it in a freeze dryer for 72 h.

5. The application according to claim 1, characterized in that, In step (3), the settling temperature is 4℃ and the time is 5h; the centrifugation speed is 5000 r / min and the time is 3min.

6. The application according to claim 1, characterized in that, In step (4), the concentration of methanol is 50 wt%; the concentration of the solution is 0.84 g / mL.

7. The application according to claim 1, characterized in that, The concentration of the secondary metabolite of Penicillium pr1 used is 50 ng / mL.

8. The application according to claim 1, characterized in that, The plant in question is rice, and the bacterial disease is bacterial blight caused by Xanthomonas.

9. The application of the active ingredient of the secondary metabolite of Penicillium pr1 as described in any one of claims 1 to 8 in the prevention and control of bacterial plant diseases, characterized in that, The active ingredient is ethyl-α-D-glucoside or ethyl-β-D-glucopyranoside.

10. The application according to claim 9, characterized in that, The concentration of the ethyl-α-D-glucoside used is 50 ng / mL; the concentration of the ethyl-β-D-glucopyranoside used is 10 ng / mL; the plant is rice; and the bacterial disease is bacterial blight caused by Xanthomonas.

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