Nano bactericide as well as preparation method and application thereof

Abstract

The invention provides a nano bactericide as well as a preparation method and application thereof. The magnolol / ZIF-8 nano composite material Magat ZIF-8 synthesized on the basis of a competitive coordination strategy simultaneously contains two coordination structures of zinc ion-2-methylimidazole and zinc ion-magnolol, so that the inhibition effect of the ZIF-8 on pathogenic fungi is remarkably improved. Besides, a compound synergistic experiment result shows that a synergistic effect exists between the MagatZIF-8 and the bactericide fludioxonil, and a new thought is provided for reduction and synergism of the bactericide.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a nano bactericide and a preparation method and application thereof. Background Art

[0002] Metal-organic frameworks (MOFs) are a new type of porous crystalline material with a periodic network structure formed by self-assembly of metal ions and organic ligands through coordination bonds. They are often used in the construction of nano drug delivery systems due to their large specific surface area, adjustable pore size and easy surface functionalization. In addition to being used as nanocarriers, MOFs' good antibacterial activity and unique antibacterial mechanism have also attracted the attention of many researchers. To date, a variety of MOFs (such as MOF-5, zeolite imidazole framework-8 (ZIF-8) and Material Institute Lavoisier-100 (MIL-100)) have been developed as non-carrier nano-fungicides to inhibit the growth of pathogens and improve the protection of hosts. ZIF-8, a widely used MOF material in biomedicine, is composed of zinc ions and 2-methylimidazole connected by coordination bonds. It can release zinc ions under acidic conditions to inhibit the growth and germination of pathogens. ZIF-8 shows good inhibitory effects on pathogenic bacteria such as Escherichia coli and Staphylococcus aureus, and has great potential for development as a commercial fungicide. However, the low biological activity of ZIF-8 against pathogenic fungi limits its application in fungal disease prevention and control. Magnolia officinalis is a natural product extracted from the stem bark of Magnolia officinalis, a traditional Chinese medicine, which shows broad-spectrum antibacterial activity and has the potential to increase the inhibitory effect of ZIF-8 on pathogenic fungi.

[0003] At present, since pathogenic fungi are much less sensitive to the antibacterial effects mediated by metal ion release than pathogenic bacteria, the research and application of antibacterial MOFs are more focused on the field of bacterial diseases. To address this problem, researchers mainly introduce another antibacterial metal ion into MOFs through the strategy of constructing bimetallic MOFs to enhance their antibacterial activity, but this method does not fundamentally change the way antibacterial MOFs act on pathogens. Summary of the invention

[0004] The purpose of the present invention is to provide a novel nano bactericide and a preparation method and application thereof.

[0005] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a nano bactericide, which is prepared by adding magnolol during the preparation process of the metal organic framework ZIF-8.

[0006] In a second aspect, the present invention provides a method for preparing the nano fungicide, which adopts a one-pot method to synthesize the nano fungicide: magnolol is added to a mixed aqueous solution of zinc acetate dihydrate and 2-methylimidazole, the precipitate is collected by magnetic stirring and centrifugation, the precipitate is washed with anhydrous ethanol and deionized water in turn to remove unreacted reactants, and finally dried and ground to obtain the nano fungicide.

[0007] In the present invention, zinc acetate dihydrate may be replaced with zinc nitrate hexahydrate.

[0008] The preparation method comprises the following steps: (1) Prepare 25 mL of 60-80 mg / mL zinc acetate dihydrate aqueous solution, 25 mL of 200-300 mg / mL 2-methylimidazole aqueous solution, and 5 mL of 50-80 mg / mL magnolol ethanol solution respectively; (2) Adding zinc acetate dihydrate aqueous solution dropwise to the 2-methylimidazole aqueous solution, and when the amount reaches 10-20 mL, adding magnolol ethanol solution dropwise to the mixed solution of zinc acetate dihydrate and 2-methylimidazole, and then adding the remaining zinc acetate dihydrate aqueous solution dropwise, stirring the reaction system with a magnetic stirrer during the entire dropping process; (3) After all reactants have been added, continue to stir the reaction system continuously with a magnetic stirrer for 1-2 hours; (4) After stirring, collect the precipitate by centrifugation, and wash the precipitate with anhydrous ethanol and deionized water in sequence for 3-5 times in total; (5) After the washed precipitate is dried in an oven, it is fully ground in a mortar to obtain a nano-fungicide powder (Mag@ZIF-8), which is then stored in a 1.5 mL centrifuge tube for later use.

[0009] Preferably, the conditions for magnetic stirring in steps (2) and (3) are: 20-25° C., 600-800 rpm.

[0010] Preferably, the centrifugation conditions in step (4) are: 10000-12000 rpm, 3-5 minutes.

[0011] Preferably, the drying conditions in step (5) are: 60-70° C., 12-16 hours.

[0012] In a specific embodiment of the present invention, the preparation method of the nano fungicide comprises the following steps: (1) Prepare 25 mL of 60 mg / mL zinc acetate dihydrate aqueous solution, 25 mL of 224 mg / mL 2-methylimidazole aqueous solution, and 5 mL of 60 mg / mL magnolol ethanol solution respectively; (2) Add zinc acetate dihydrate aqueous solution dropwise to the 2-methylimidazole aqueous solution. When the amount reaches 12.5 mL, add magnolol ethanol solution dropwise to the mixed solution of zinc acetate dihydrate and 2-methylimidazole. Then, add the remaining 12.5 mL of zinc acetate dihydrate aqueous solution dropwise. During the entire addition process, stir the reaction system with a magnetic stirrer. (3) After all reactants have been added, the reaction system is continuously stirred by a magnetic stirrer for 2 hours; (4) After stirring, collect the precipitate by centrifugation, and wash the precipitate with anhydrous ethanol and deionized water in sequence for a total of 3 times; (5) The washed precipitate is dried and then ground to obtain nano fungicide powder.

[0013] In a third aspect, the present invention provides a compound fungicide, the active ingredients of which are the nano fungicide and fludioxonil.

[0014] Preferably, the mass ratio of the nano fungicide to fludioxonil is 1:1.

[0015] In a fourth aspect, the present invention provides any of the following applications of the nano bactericide or the compound bactericide or a material containing the nano bactericide or the compound bactericide: 1) Used to prepare broad-spectrum antibacterial agents; 2) Used to inhibit pathogenic fungi; 3) Used for fungal disease prevention and control; 4) Used to develop new antibacterial materials.

[0016] In the present invention, the fungus includes but is not limited to Botrytis cinerea Botrytis cinerea graminearum ( Fusarium graminearum )、Sheath blight fungus( Rhizoctonia solani ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Colletotrichum oxysporum ( Colletotrichum acutatum ).

[0017] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The magnolol / ZIF-8 nanocomposite (Mag@ZIF-8) synthesized based on the competitive coordination strategy of the present invention contains two coordination structures, zinc ion-2-methylimidazole and zinc ion-magnoliol, which significantly increases the inhibitory effect of ZIF-8 on pathogenic fungi. The results of the bioassay showed that the median effective concentration (EC) of ZIF-8 on the growth of gray mold mycelium was 50) was 886.1 μg / mL, while the EC of Mag@ZIF-8 on the mycelial growth of Botrytis cinerea 50 The value was 68.3 μg / mL. This indicates that under the same experimental conditions, the inhibitory activity of Mag@ZIF-8 on pathogenic fungi is higher than that of ZIF-8, and that the addition of magnolol significantly improves the inhibitory effect of ZIF-8 on pathogenic fungi.

[0018] In addition, the results of the compound synergistic experiment showed that the actual inhibition rate (67.8%) of the binary mixture of Mag@ZIF-8 and the fungicide fludioxonil (1:1) on the mycelial growth of Botrytis cinerea was higher than its theoretical inhibition rate (57.1%), indicating that there is a synergistic effect between Mag@ZIF-8 and fludioxonil, which provides a new idea for reducing the dosage of fungicides and increasing their efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Characterization of ZIF-8 and Mag@ZIF-8 in the preferred embodiments of the present invention. (A) Transmission electron microscope images of ZIF-8 and Mag@ZIF-8. (B) Particle size distribution of ZIF-8 and Mag@ZIF-8. (C) Macroscopic morphology of ZIF-8 and Mag@ZIF-8 suspensions.

[0020] Figure 2 The figure shows the inhibitory effect of ZIF-8 (A) and Mag@ZIF-8 (B) on the mycelial growth of Botrytis cinerea in the preferred embodiments of the present invention.

[0021] Figure 3 The antibacterial activity of Mag@ZIF-8 in the preferred embodiment of the present invention against pathogenic fungi such as Fusarium graminearum (A), Rhizoctonia solani (B), Sclerotinia sclerotiorum (C), Colletotrichum gloeosporioides (D), and Colletotrichum oxysporum (E).

[0022] Figure 4 This is the synergistic effect between Mag@ZIF-8 and common fungicides in the preferred embodiment of the present invention. DETAILED DESCRIPTION

[0023] The present invention aims to provide a nano fungicide and a synthesis method thereof, as well as application thereof in the prevention and control of fungal diseases.

[0024] The present invention adopts the following technical solution: The present invention adopts a one-pot method to synthesize the nano fungicide Mag@ZIF-8. Specifically, magnolol is added to a mixed aqueous solution of zinc acetate dihydrate and 2-methylimidazole, a precipitate is obtained by magnetic stirring and centrifugation, and then the unreacted reactants are removed by washing with anhydrous ethanol and deionized water, and finally Mag@ZIF-8 powder is obtained by drying and grinding. Mag@ZIF-8 is a nanosphere with an average particle size of 148.7 nm. The Mag@ZIF-8 aqueous suspension is khaki.

[0025] The inhibitory effect of Mag@ZIF-8 on the mycelial growth of Botrytis cinerea is better than that of ZIF-8, and Mag@ZIF-8 can be compounded with the fungicide fludioxonil to enhance the synergy.

[0026] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0027] The gray mold fungus B05.10 used in the following examples was provided by Professor Hou Yiping of the College of Plant Protection, Nanjing Agricultural University. The strain B05.10 can be found in Bian, C., Duan, Y., Wang, J., Xiu, Q., Wang, J., Hou, Y., ...&Zhou, M. (2020). Validamycin A induces broad-spectrum resistance involving salicylic acid and jasmonic acid / ethylene signaling pathways. Molecular Plant-Microbe Interactions, 33(12), 1424-1437. Fusarium graminearum (PH-1), Sclerotinia sclerotiorum (YD5), Colletotrichum gloeosporioides (LN-48), and Colletotrichum oxysporum (HTTJ1) were kindly provided by Professor Liu Feng from the College of Plant Protection, Shandong Agricultural University, and Rhizoctonia solani (AG4) was kindly provided by Professor Liu Wende from the Institute of Plant Protection, Chinese Academy of Agricultural Sciences. For Fusarium graminearum, see Ma, D., Wang, G., Zhu, J., Mu, W., Dou, D.,&Liu, F. (2022). Green leaf volatile trans-2-hexenal inhibits the growth of Fusarium graminearumby inducing membrane damage, ROS accumulation, and cell dysfunction. Journal of Agricultural and Food Chemistry, 70(18), 5646-5657. For the study of sheath blight pathogens, see Zhang, Z., Xia, X., Du, Q., Xia, L., Ma, X., Li, Q., & Liu, W. (2021). Genome sequence of Rhizoctonia solani anastomosis group 4 strainRhs4ca, a widespread pathomycete in field crops. Molecular Plant-MicrobeInteractions, 34(7), 826-829. For Sclerotinia sclerotiorum, see Huang, X., Luo, J., Li, B, Song, Y, Mu, W., & Liu, F. (2019). Bioactivity, physiological characteristics and efficacy of the SDHI fungicide pydiflumetofen against Sclerotinia sclerotiorum . Pesticide Biochemistry and Physiology, 160, 70-78. For Colletotrichum gloeosporioides and Colletotrichum oxysporum, see He, L., Li, X., Gao, Y., Li, B., Mu, W.,&Liu, F. (2019).Characterization and fungicide sensitivity of Colletotrichum spp. from different hosts in Shandong, China. Plant Disease, 103(1), 34-43. The magnolol used in the following examples was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with the item number M813634, and the fludioxonil was purchased from Shanghai MacLean Biochemical Technology Co., Ltd. with the item number F980645.

[0028] Example 1 Synthesis of ZIF-8 and Mag@ZIF-8 Mag@ZIF-8 was synthesized by a one-pot method. The specific steps are as follows: (1) Prepare zinc acetate dihydrate aqueous solution (25 mL, 60 mg / mL), 2-methylimidazole aqueous solution (25 mL, 224 mg / mL), and magnolol ethanol solution (5 mL, 60 mg / mL) respectively; (2) Add zinc acetate dihydrate aqueous solution dropwise into the 2-methylimidazole aqueous solution through a rubber-tipped dropper. When half of the solution (12.5 mL) is added, add magnolol ethanol solution dropwise into the mixture of zinc acetate dihydrate and 2-methylimidazole. Then, add the remaining zinc acetate dihydrate aqueous solution (12.5 mL) dropwise. During the entire addition process, stir the reaction system with a magnetic stirrer (25°C, 600 rpm). (3) After all reactants have been added, the reaction system was continuously stirred using a magnetic stirrer (25 °C, 600 rpm) for 2 h. (4) After stirring, the reaction product was centrifuged (10,000 rpm, 3 minutes), and the obtained precipitate was washed with anhydrous ethanol and deionized water to remove unreacted reactants, for a total of three times; (5) The washed precipitate was dried in an oven (60 °C, 12 h), and then ground in a mortar to obtain Mag@ZIF-8 powder, which was stored in a 1.5 mL centrifuge tube for later use.

[0029] The synthesis steps of ZIF-8 were the same as those of Mag@ZIF-8, except that magnolol was not added.

[0030] Example 2 Characterization of ZIF-8 and Mag@ZIF-8 An appropriate amount of Mag@ZIF-8 powder was added to a centrifuge tube filled with deionized water to prepare a 250 μg / mL suspension. Then, the suspension was placed in an ultrasonic crusher (power 500 W, 30 minutes) for ultrasonication to uniformly disperse Mag@ZIF-8 in deionized water. The preparation of ZIF-8 suspension also refers to the above method.

[0031] 5 μL Mag@ZIF-8 suspension was pipetted with a pipette and dropped onto the grid. The nanoparticles were naturally air-dried at room temperature for 12 hours to fix the nanoparticles on the grid. The prepared samples were stored in a desiccator to prevent the samples from getting wet or contaminated. The microscopic morphology of Mag@ZIF-8 was observed and photographed using a transmission electron microscope (model: Tecnai G2 F30, FEI Company, USA). The particle size of the nanoparticles in the transmission electron microscope photos was counted using ImageJ software (version: 1.52a, National Institutes of Health, USA), and the particle size distribution of the nanoparticles was analyzed using GraphPad software (version: 8.0.2, GraphPad Software, USA). ZIF-8 was used as a control. The results showed that ZIF-8 nanoparticles were polyhedral with an average particle size of 209.1 nm; Mag@ZIF-8 nanoparticles were nearly spherical with an average particle size of 148.7 nm ( Figure 1 ).

[0032] 1.5 mL of Mag@ZIF-8 suspension was pipetted into a sample vial, and the macroscopic state of the suspension was observed and photographed using a digital camera (model: EOS R100, Canon, Japan). The ZIF-8 suspension was used as a control. The results showed that the ZIF-8 suspension was white, while the Mag@ZIF-8 suspension was earthy yellow ( Figure 1 ).

[0033] Example 3 Inhibitory effect of ZIF-8 and Mag@ZIF-8 on hyphae growth of Botrytis cinerea The inhibitory effect of ZIF-8 and Mag@ZIF-8 on the mycelial growth of Botrytis cinerea was determined by the mycelial growth rate method. The specific steps are as follows: (1) Add an appropriate amount of Mag@ZIF-8 powder into a centrifuge tube filled with sterile deionized water to prepare a 5000 μg / mL stock solution. After ultrasonication for 30 minutes to fully disperse the nanoparticles, dilute the stock solution with deionized water to 2500, 1250, 625, and 312.5 μg / mL, respectively. The preparation of the ZIF-8 test solution also refers to the above method; (2) Add appropriate amount of Mag@ZIF-8 suspension to potato dextrose agar (PDA) medium precooled to about 50°C to make the final concentration reach 0, 31.25, 62.5, 125, 250 and 500 μg / mL, vortex mix and pour into a disposable plastic culture dish (15 mL / dish), dry and inoculate the Botrytis cinerea (strain B05.10) cake (7 mm in diameter) with the mycelium side facing down. Use the same concentration of ZIF-8 as a control; (3) After inoculation, place the culture dish in the dark at 25°C for incubation; (4) After 2 days of culture, the colony expansion diameter was measured by the cross method to calculate the inhibition rate, and the test concentration and inhibition rate were converted to calculate the EC using DPS software (version: v9.05, Hangzhou Ruifeng Information Technology Co., Ltd.). 50 value.

[0034] The results showed that ZIF-8 had an EC 50 The value was 886.1 μg / mL, while the EC value of Mag@ZIF-8 on the mycelial growth of Botrytis cinerea was 50 The value was 68.3 μg / mL, indicating that the inhibitory activity of Mag@ZIF-8 against Botrytis cinerea was significantly higher than that of ZIF-8 ( Figure 2 ).

[0035] Example 4 Antibacterial activity of Mag@ZIF-8 against pathogenic fungi such as Fusarium graminearum The mycelium growth rate method was used to determine the inhibitory effect of Mag@ZIF-8 on the mycelium growth of five pathogenic fungi, including Fusarium graminearum. The specific steps are as follows: (1) Mag@ZIF-8 suspensions with concentrations of 312.5, 625, 1250, 2500, and 5000 μg / mL were prepared in deionized water; (2) Add an appropriate amount of Mag@ZIF-8 suspension to the PDA medium precooled to about 50°C to make the final concentrations of 0, 31.25, 62.5, 125, 250 and 500 μg / mL, mix well and pour into a plastic culture dish; (3) Inoculate the dried culture dish with a fungal cake (7 mm in diameter) of pathogenic fungi (Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum, Colletotrichum gloeosporioides or Colletotrichum oxysporum), and transfer the inoculated culture dish to 25°C in the dark for cultivation; (4) After 2-5 days of culture, the colony expansion diameter was measured by the cross method to calculate the EC of Mag@ZIF-8 for inhibiting mycelial growth. 50 value.

[0036] The results showed that Mag@ZIF-8 exhibited good inhibitory activity against Fusarium graminearum, Rhizoctonia solani, Sclerotinia sclerotiorum, Colletotrichum gloeosporioides and Colletotrichum oxysporum. 50 The values ​​were 113.6, 148.1, 103.1, 107.5 and 207.8 μg / mL ( Figure 3 ).

[0037] Example 5 Evaluation of the synergistic effect of the combination of Mag@ZIF-8 and fungicides The synergistic effect of Mag@ZIF-8 and 8 commonly used fungicides was evaluated by a simple mixing method. The specific steps are as follows: (1) Prepare the test solutions of Mag@ZIF-8 (2500 μg / mL), carbendazim (40 μg / mL), boscalid (25 μg / mL), pyraclostrobin (2 μg / mL), propiconazole (0.5 μg / mL), procymidone (2.5 μg / mL), fludioxonil (0.05 μg / mL), iprodione (1 μg / mL), and pyrimethanil (0.5 μg / mL) with sterile deionized water; (2) Add Mag@ZIF-8 (1.5 mL) and a single fungicide (1.5 mL) to the PDA medium (15 mL) precooled to about 50°C, vortex mix, and pour into a plastic culture dish. Use a PDA plate containing only deionized water, Mag@ZIF-8, or fungicide as a control; (3) Inoculate the dried culture dish with a cake of Botrytis cinerea (strain B05.10) (7 mm in diameter), and transfer the inoculated culture dish to 25°C in the dark for cultivation; (4) After 2 days of cultivation, the colony expansion diameter was measured by the cross method to calculate the actual combined inhibition rate of the binary mixture of Mag@ZIF-8 and fungicide. The theoretical combined inhibition rate of the binary mixture of Mag@ZIF-8 and fungicide = the inhibition rate of Mag@ZIF-8 + the inhibition rate of fungicide. If the actual combined inhibition rate of the binary mixture of Mag@ZIF-8 and fungicide is higher / equal to / lower than its theoretical combined inhibition rate, it indicates that there is a synergistic / additive / antagonistic effect between the two.

[0038] The results showed that Mag@ZIF-8 had a synergistic effect with fludioxonil (actual combined inhibition rate 67.8%>theoretical combined inhibition rate 57.1%), an additive effect with carbendazim (actual combined inhibition rate 30.8%=theoretical combined inhibition rate 30.8%), and an antagonistic effect with boscalid (actual combined inhibition rate 50.9%<theoretical combined inhibition rate 69.6%), azoxystrobin (actual combined inhibition rate 38.8%<theoretical combined inhibition rate 54.5%), propiconazole (actual combined inhibition rate 49.1%<theoretical combined inhibition rate 60.3%), procymidone (actual combined inhibition rate 32.1%<theoretical combined inhibition rate 50.4%), iprodione (actual combined inhibition rate 32.1%<theoretical combined inhibition rate 50.4%) and pyrimethanil (actual combined inhibition rate 33.5%<theoretical combined inhibition rate 39.7%). Figure 4 ).

[0039] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A nano fungicide, characterized in that: It is prepared by adding magnolol during the preparation of the metal organic framework ZIF-8.

2. The method for preparing the nano fungicide according to claim 1, characterized in that: The nano fungicide is synthesized by a one-pot method: magnolol is added to a mixed aqueous solution of zinc acetate dihydrate and 2-methylimidazole, the precipitate is collected by magnetic stirring and centrifugation, the precipitate is washed with anhydrous ethanol and deionized water in turn, and finally dried and ground to obtain the nano fungicide.

3. The method according to claim 2, characterized in that The following steps are involved: (1) Prepare 25 mL of 60-100 mg / mL zinc acetate dihydrate aqueous solution, 25 mL of 200-300 mg / mL 2-methylimidazole aqueous solution, and 5 mL of 50-80 mg / mL magnolol ethanol solution respectively; (2) Adding zinc acetate dihydrate aqueous solution dropwise to the 2-methylimidazole aqueous solution, and when the amount reaches 10-20 mL, adding magnolol ethanol solution dropwise to the mixed solution of zinc acetate dihydrate and 2-methylimidazole, and then adding the remaining zinc acetate dihydrate aqueous solution dropwise, stirring the reaction system with a magnetic stirrer during the entire dropping process; (3) After all reactants have been added, continue to stir the reaction system continuously with a magnetic stirrer for 1-2 hours; (4) After stirring, collect the precipitate by centrifugation, and wash the precipitate with anhydrous ethanol and deionized water in sequence for 3-5 times in total; (5) The washed precipitate is dried and then ground to obtain nano fungicide powder.

4. The method according to claim 3, characterized in that The conditions for magnetic stirring in steps (2) and (3) are: 20-25°C, 600-800 rpm.

5. The method according to claim 3, characterized in that: The centrifugation conditions in step (4) are: 10000-12000 rpm, 3-5 minutes.

6. The method according to claim 3, characterized in that The drying conditions in step (5) are: 60-70°C, 12-16 hours.

7. A compound fungicide, characterized in that: The active ingredients are the nano fungicide according to claim 1 and fludioxonil.

8. The compound fungicide according to claim 7, characterized in that: The mass ratio of the nano fungicide to fludioxonil is 1:

1.

9. Any of the following uses of the nano bactericide according to claim 1 or the compound bactericide according to claim 7 or 8 or a material containing the nano bactericide or the compound bactericide: 1) Used to prepare broad-spectrum antibacterial agents; 2) Used to inhibit pathogenic fungi; 3) Used for fungal disease prevention and control; 4) Used to develop new antibacterial materials.

10. The use according to claim 9, characterized in that: The fungi include Botrytis cinerea ( Botrytis cinerea graminearum ( Fusarium graminearum )、Sheath blight pathogen ( Rhizoctonia solani ), Sclerotinia sclerotiorum ( Sclerotinia sclerotiorum ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Colletotrichum oxysporum ( Colletotrichum acutatum ).

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