CN110558336A - Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof - Google Patents
Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof Download PDFInfo
- Publication number
- CN110558336A CN110558336A CN201910652482.1A CN201910652482A CN110558336A CN 110558336 A CN110558336 A CN 110558336A CN 201910652482 A CN201910652482 A CN 201910652482A CN 110558336 A CN110558336 A CN 110558336A
- Authority
- CN
- China
- Prior art keywords
- ginkgo
- extract
- lettuce
- parts
- ethanol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/36—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids
- A01N37/38—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system
- A01N37/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a singly bound oxygen or sulfur atom attached to the same carbon skeleton, this oxygen or sulfur atom not being a member of a carboxylic group or of a thio analogue, or of a derivative thereof, e.g. hydroxy-carboxylic acids having at least one oxygen or sulfur atom attached to an aromatic ring system having at least one carboxylic group or a thio analogue, or a derivative thereof, and one oxygen or sulfur atom attached to the same aromatic ring system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
Abstract
The invention belongs to the technical field of biological control, and particularly relates to a biocontrol agent for controlling lettuce sclerotinia rot and a preparation method thereof. The biocontrol preparation comprises the following components in parts by weight: 15-35 parts of endophytic fungus spirillum roseum fermentation liquor of ginkgo, 10-20 parts of ginkgo leaf extract and 15-25 parts of ginkgo episperm extract; and (3) mixing fermentation liquor, a ginkgo leaf extract and a ginkgo episperm extract which are prepared by carrying out solid culture and liquid culture on the spirosporium roseum according to the parts by weight to obtain the biocontrol preparation. According to the invention, the spirospora rosea fermentation liquor, the ginkgo leaf extract and the ginkgo episperm extract are adopted as active ingredients to be compounded according to a certain proportion, so that the control effect can be obviously improved compared with a single ingredient, the growth of lettuce seedlings can be promoted, the lettuce sclerotiniose can be obviously inhibited, the technical problems of easy generation of drug resistance and environmental harm in chemical control can be avoided, and the application prospect is certain.
Description
Technical Field
the invention belongs to the technical field of biological control, and particularly relates to a biocontrol agent for controlling lettuce sclerotinia rot and a preparation method thereof.
background
Lettuce is an important vegetable crop in China, sclerotinia is a common disease on lettuce, the pathogenic bacteria of the sclerotinia is sclerotinia sclerotiorum, and sclerotinia sclerotiorum caused by the pathogenic bacteria can be caused in successive years once the sclerotinia sclerotiorum is attacked, so that the yield and the quality of the lettuce are seriously reduced, and economic loss is caused. The sclerotinia sclerotiorum has wide host range, the sclerotium formed on the disease residues has strong stress resistance, the ascospore can be diffused and spread in a short distance along with the airflow in the field, and the characteristics cause that the control of the sclerotinia sclerotiorum is very difficult. For the prevention and treatment of sclerotinia, no really economic and effective prevention and treatment measures exist at present, and the prevention and treatment mainly depend on chemical pesticides and cultivation measures in actual production.
The bactericide for preventing and treating sclerotinia rot is prepared with carbendazim, benralin, procymidone and other single preparation and over twenty kinds of compounded preparation, but has inconvenient application, long effective protection period due to the diffusion of ascospores from sclerotinia sclerotiorum, and limited pesticide application due to the occurrence of the problems of drug resistance of sclerotinia sclerotiorum and harm of pesticide residue to environment, human and livestock. Among the cultivation control measures, the rotation of paddy and dry land is the most effective disease prevention measure for reducing the initial infection source, but the rotation of paddy and dry land only reduces the local bacterial source and cannot prevent the foreign bacterial source spread by wind, so the prevention effect is often unstable by the measure alone. Although effective, the method for breeding and utilizing the disease-resistant variety to prevent and treat the lettuce sclerotinia rot is not capable of finding the real disease-resistant variety or resource, on the one hand, the work is still in the laboratory research stage at present, and the application in production needs a period of time. In the biological control research, the Sporotrichum roseum can parasitize sclerotinia fungi and has a certain inhibiting effect on sclerotinia sclerotiorum diseases, but the Sporotrichum roseum cannot be popularized and used in a large area, and the problems of poor production performance, unstable control effect caused by various complex factors and the like often exist in the practical production application.
disclosure of Invention
aiming at the technical problems, the invention aims to provide a biocontrol agent for preventing and treating lettuce sclerotinia rot and a preparation and use method thereof.
in order to achieve the purpose, the invention adopts the following technical scheme:
Firstly, the invention provides a biocontrol agent for preventing and treating lettuce sclerotinia rot, which comprises the following components in parts by weight: 15-35 parts of endophytic fungus spirillum roseum fermentation liquid, 10-20 parts of ginkgo biloba extract and 15-25 parts of ginkgo biloba exocarp extract.
preferably, the biocontrol agent comprises the following components in parts by weight: 25 parts of endophytic fungus pinopillaria rosea fermentation liquor, 15 parts of ginkgo leaf extract and 20 parts of ginkgo episperm extract.
Secondly, the invention provides a preparation method of the biocontrol agent for preventing and treating lettuce sclerotinia rot, which comprises the following steps:
(1) Inoculating activated and cultured spirillum roseum to the center of a PDA flat plate, placing the PDA flat plate in a constant-temperature incubator at 28 ℃ for culturing for 2-3d, then inoculating a purified cake of spirillum roseum to a PDB liquid culture medium, and culturing for 2-3d on a constant-temperature shaking table to obtain the spirillum roseum fermentation liquor;
(2) Drying folium Ginkgo, pulverizing, sieving with 40-60 mesh sieve, placing into an extraction tank, simultaneously adding 60-80% ethanol, extracting for 2-3 times (each time for 60-70 min), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5-1/10 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15 hr, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1-2h each time for 2-3 times, wherein the material-liquid ratio is 1:6-12(W/V), filtering the decoctions, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution to a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to make the final concentration of the ethanol reach 60-80% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, standing, washing the precipitate with 80-90% ethanol for 3-5 times, and vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) And uniformly mixing the fusarium oxysporum fermentation liquid, the ginkgo leaf extract and the ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
Further, the temperature of constant temperature shaking table culture in the step (1) is 28-30 ℃, and the rotating speed is 200-220 r/min.
further, the thallus concentration of the spirillum roseum in the fermentation liquor of the spirillum roseum obtained in the step (1) is more than or equal to 1 × 107cfu/ml。
Further, the dosage of the ethanol in the extraction tank in the step (2) is 4-8 times of the weight of the ginkgo leaves.
Further, the ethanol containing acetic acid in the step (2) is composed of ethanol with the volume concentration of 80-90% and acetic acid with the volume percentage of 0.1-0.5%.
Further, the stirring temperature of the magnetic stirrer in the step (3) is 20-30 ℃, and the rotating speed is 1400-1800 r/min.
Finally, the invention provides a using method of the biocontrol agent for preventing and treating lettuce sclerotinia rot, which comprises the following steps: after the lettuce seeds are sowed, taking the biocontrol agent according to 10-20ml per hundred seeds, diluting the biocontrol agent by 10-20 times with sterile water, and then watering and applying a seedbed; after 5-6 leaves grow on the lettuce seedlings, the biocontrol agent is diluted by 20-40 times by sterile water and is uniformly sprayed on the surfaces of the leaves of the lettuce seedling plants by a sprayer.
The two using methods of the biocontrol agent for preventing and treating the lettuce sclerotinia rot can be used alternatively or in combination.
compared with the prior art, the invention has the following beneficial effects:
(1) The spirosporium roseum is slow in growth and reproduction, and can obviously improve the growth speed of thalli after being compounded with the ginkgo leaf extract and the ginkgo episperm extract according to a certain proportion, so that the competition effect of the spirosporium roseum and sclerotinia on the survival space and nutrition is promoted, and the infection of the sclerotinia on lettuce is reduced; meanwhile, the growth of hypha of the spirillum roseum can be promoted, and the infection parasitic rate of the hypha to sclerotinia sclerotiorum is improved. In addition, the activity of cell wall degrading enzyme secreted by the spirillum roseum is improved, the cell wall of the sclerotinia sclerotiorum can be effectively degraded to cause the sclerotinia sclerotiorum to break and die, and the inhibition effect on the lettuce sclerotiniose is realized.
(2) The ginkgo biloba extract and the ginkgo biloba testa extract contain abundant ginkgolic acid and flavonoid compounds, wherein the alkyl side chain of the ginkgolic acid plays an important role in bacteriostasis. The chemical structures of flavonoids in the ginkgo biloba extract and the antibiotic generated by the endophytic fungus spirosporium roseum have complementarity, and partial sugar groups of the flavonoid oxazeolone C-glucose play a role of modifying groups on the structure of the antibiotic, so that the ginkgo biloba extract has a synergistic antibacterial effect. In addition, the antibiotic produced by the gingko endophytic fungus spirillum roseum plays a role in inhibiting the cell wall synthesis of pathogenic bacteria; after the interaction between the flavonoid compounds in the ginkgo leaf extract and the phospholipid hydrophilic area on the cell membrane, the arrangement disorder of phospholipid molecules on the cell membrane of pathogenic bacteria can be caused, and the metabolic process of nutrition and energy of pathogenic bacteria cells can be influenced. Therefore, the action targets of the endophytic fungus spirillum roseum and the metabolite thereof are different from those of the ginkgo biloba extract and the ginkgo biloba testa extract, the endophytic fungus spirillum roseum and the metabolite thereof have the action of inhibiting the synthesis of germ cell walls, the ginkgo biloba testa extract has the action of directly killing lettuce sclerotinia sclerotiorum through the strong oxidizing action of phenolic acid, and the flavonoid compounds in the ginkgo biloba extract and the antibiotic generated by the endophytic fungus spirillum roseum have the synergistic bacteriostasis. The compounding of the three components can enhance the prevention and treatment effect on the lettuce sclerotinia sclerotiorum and reduce the resistance of the lettuce sclerotinia sclerotiorum on a biocontrol agent.
(3) After sowing, the biocontrol preparation is applied to the seedbed, so that the germination of sclerotia remained in soil can be obviously inhibited, the growth and development of lettuce seedlings can be promoted, and the disease resistance of the lettuce seedlings is improved. After 5-6 leaves grow out from seedlings, the biocontrol agent disclosed by the invention is sprayed on the leaves, so that the expansion and infection of sclerotinia sclerotiorum hyphae can be obviously inhibited, the incidence rate of the lettuce sclerotinia sclerotiorum disease is reduced, and the two methods can be used alternatively or in combination, so that the combined use effect is better. Compared with the conventional chemical control, the biocontrol agent is more environment-friendly and is not easy to generate drug resistance.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The endophytic fungus Pink spirillum used in the following examples was purchased from the institute of Biotechnology, Beijing Beinanna, and the ginkgo leaves and the ginkgo testa were collected by themselves, and other used chemical instruments and medicines were purchased from conventional methods.
Example 1
A preparation method of a biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps:
(1) Inoculating activated and cultured Spirosporium roseum to the center of a PDA (PDA) plate, placing the PDA plate in a constant-temperature incubator for culturing for 2-3d, then inoculating the purified cake of Spirosporium roseum to a PDB (plant protein B) liquid culture medium, and culturing for 2-3d at 220r/min and 30 ℃ on a constant-temperature shaking bed to obtain the Spirosporium roseum fermentation liquid, wherein the concentration of the Spirosporium roseum is more than or equal to 1 x 107cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 40 mesh sieve, placing into an extraction tank, simultaneously adding 70% ethanol 8 times of folium Ginkgo weight, extracting for 3 times (60 min each time), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of ethanol with the volume concentration of 90% and acetic acid with the volume percentage of 0.5%;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 2 hours each time for 2 times, wherein the material-liquid ratio is 1:10(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 80% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 25 ℃, the rotating speed is 1600r/min, after standing, washing the precipitate with ethanol with the volume concentration of 90% for 3-5 times, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) Uniformly mixing 25 parts of endophytic fungus pinosylvia rosea fermentation liquor, 15 parts of ginkgo leaf extract and 20 parts of ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
The application method of the biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps: after the lettuce seeds are sowed, taking the biocontrol agent according to 10ml per hundred seeds, diluting the biocontrol agent by 15 times with sterile water, and then watering a seedbed; meanwhile, after 5-6 leaves grow on the lettuce seedlings, the biocontrol agent is diluted by 30 times by using sterile water and is uniformly sprayed on the surfaces of the leaves of the lettuce seedlings by using a sprayer.
Example 2
A preparation method of a biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps:
(1) Inoculating activated Spirosporium roseum to the center of a PDA plate, culturing in a constant temperature incubator at 28 deg.C for 2-3d, inoculating the purified cake of Spirosporium roseum to PDB liquid culture medium, and culturing in a PDB liquid culture mediumCulturing at 28 deg.C and 200r/min for 2-3d to obtain the fermentation liquid of Spirosporium roseum, wherein the thallus concentration of Spirosporium roseum is not less than 1 × 107cfu/ml；
(2) drying folium Ginkgo, pulverizing, sieving with 50 mesh sieve, placing into an extraction tank, simultaneously adding 60% ethanol 6 times of folium Ginkgo weight, extracting for 70min for 2 times, and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/10 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of ethanol with the volume concentration of 90% and acetic acid with the volume percentage of 0.1%;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1h each time for 3 times, wherein the material-liquid ratio is 1:8(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 70% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 30 ℃, the rotating speed is 1400r/min, washing the precipitate with ethanol with the volume concentration of 80% for 3-5 times after standing, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) Uniformly mixing 30 parts of endophytic fungus pinosylvia rosea fermentation liquor, 10 parts of ginkgo leaf extract and 20 parts of ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
the application method of the biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps: after the lettuce seeds are sowed, taking the biocontrol agent according to 15ml per hundred seeds, diluting the biocontrol agent by 20 times with sterile water, and then watering a seedbed; meanwhile, after 5-6 leaves grow on the lettuce seedlings, the biocontrol agent is diluted by 40 times by using sterile water and is uniformly sprayed on the surfaces of the leaves of the lettuce seedlings by using a sprayer.
Example 3
A preparation method of a biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps:
(1) inoculating activated and cultured Spirosporium roseum to the center of a PDA (PDA) plate, placing the PDA plate in a constant-temperature incubator for culturing for 2-3d, then inoculating the purified cake of Spirosporium roseum to a PDB (plant protein B) liquid culture medium, and culturing for 2-3d at the temperature of 210r/min on a constant-temperature shaking bed at 28 ℃ to obtain the Spirosporium roseum fermentation liquid, wherein the thallus concentration of the Spirosporium roseum is more than or equal to 1 x 107cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 60 mesh sieve, placing into an extraction tank, simultaneously adding 80% ethanol 8 times of folium Ginkgo weight, extracting for 70min for 2 times, and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/7 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of 85% ethanol by volume concentration and 0.1% acetic acid by volume percentage;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 2 hours each time for 2 times, wherein the material-liquid ratio is 1:10(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 80% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 30 ℃, the rotating speed is 1800r/min, washing the precipitate with ethanol with the volume concentration of 80% for 3-5 times after standing, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) Uniformly mixing 35 parts of endophytic fungus pinosylvia rosea fermentation liquor, 10 parts of ginkgo leaf extract and 15 parts of ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
The application method of the biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps: after 5-6 leaves grow out from the lettuce seedlings, the biocontrol agent is diluted by 20 times by using sterile water and is uniformly sprayed on the leaf surfaces of the lettuce seedling plants by using a sprayer.
example 4
a preparation method of a biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps:
(1) Inoculating activated and cultured Spirosporium roseum to the center of a PDA (PDA) plate, placing the PDA plate in a constant-temperature incubator for culturing for 2-3d, then inoculating the purified cake of Spirosporium roseum to a PDB (plant protein B) liquid culture medium, and culturing for 2-3d at the temperature of 210r/min on a constant-temperature shaking bed at 30 ℃ to obtain the Spirosporium roseum fermentation liquid, wherein the thallus concentration of the Spirosporium roseum is more than or equal to 1 x 107cfu/ml；
(2) drying folium Ginkgo, pulverizing, sieving with 60 mesh sieve, placing into an extraction tank, simultaneously adding 80% ethanol 4 times of folium Ginkgo weight, extracting for 3 times (60 min each time), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of 85% ethanol by volume concentration and 0.5% acetic acid by volume percentage;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 3 times with 2h each time, wherein the material-liquid ratio is 1:12(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 60% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 20 ℃, the rotating speed is 1800r/min, washing the precipitate with ethanol with the volume concentration of 90% for 3-5 times after standing, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) And uniformly mixing 20 parts of endophytic fungus pinosylvia rosea fermentation liquor, 15 parts of ginkgo leaf extract and 25 parts of ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
The application method of the biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps: after 5-6 leaves grow out from the lettuce seedlings, the biocontrol agent is diluted by 30 times by using sterile water and is uniformly sprayed on the leaf surfaces of the lettuce seedling plants by using a sprayer.
Example 5
A preparation method of a biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps:
(1) inoculating activated and cultured Spirosporium roseum to the center of a PDA (PDA) plate, placing the PDA plate in a constant-temperature incubator for culturing for 2-3d, then inoculating the purified cake of Spirosporium roseum to a PDB (plant protein B) liquid culture medium, and culturing for 2-3d at the temperature of 200r/min on a constant-temperature shaking bed at 30 ℃ to obtain the Spirosporium roseum fermentation liquid, wherein the concentration of the Spirosporium roseum is more than or equal to 1 x 107cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 50 mesh sieve, placing into an extraction tank, simultaneously adding 70% ethanol 6 times of folium Ginkgo weight, extracting for 3 times (60 min each time), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/10 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of ethanol with the volume concentration of 80% and acetic acid with the volume percentage of 0.5%;
(3) Decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1h each time for 23 times, wherein the material-liquid ratio is 1:8(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 70% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 25 ℃, the rotating speed is 1600r/min, after standing, washing the precipitate with ethanol with the volume concentration of 90% for 3-5 times, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) uniformly mixing 25 parts of endophytic fungus pinosylvia rosea fermentation liquor, 15 parts of ginkgo leaf extract and 20 parts of ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
The application method of the biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps: after the lettuce seeds are sowed, the biocontrol agent is taken according to the volume of 20ml per hundred seeds, and the lettuce seeds are diluted by 20 times by sterile water and then applied to a seedbed.
Example 6
A preparation method of a biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps:
(1) inoculating activated and cultured Spirosporium roseum to the center of a PDA (PDA) plate, placing the PDA plate in a constant-temperature incubator for culturing for 2-3d, then inoculating the purified cake of Spirosporium roseum to a PDB (plant protein B) liquid culture medium, and culturing for 2-3d at 220r/min on a constant-temperature shaking bed at 28 ℃ to obtain the Spirosporium roseum fermentation liquid, wherein the concentration of the Spirosporium roseum is more than or equal to 1 x 107cfu/ml；
(2) Drying folium Ginkgo, pulverizing, sieving with 40 mesh sieve, placing into an extraction tank, simultaneously adding 60% ethanol 4 times of folium Ginkgo weight, extracting for 70min for 2 times, and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15h, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract; the ethanol containing acetic acid consists of ethanol with the volume concentration of 80% and acetic acid with the volume percentage of 0.1%;
(3) decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1h each time for 3 times, wherein the material-liquid ratio is 1:6(W/V), filtering the decoction liquids respectively, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution into a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to enable the final concentration of the ethanol to reach 60% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, wherein the stirring temperature is 20 ℃, the rotating speed is 1400r/min, washing the precipitate with ethanol with the volume concentration of 80% for 3-5 times after standing, and then carrying out vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) And uniformly mixing 15 parts of endophytic fungus pinosylvia rosea fermentation liquor, 20 parts of ginkgo leaf extract and 25 parts of ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
The application method of the biocontrol agent for preventing and treating lettuce sclerotinia rot comprises the following steps: after the lettuce seeds are sowed, the biocontrol agent is taken according to the volume of 10ml per hundred seeds, and is diluted by 10 times by sterile water and then applied to a seedbed.
Comparative example 1 is a blank control with only clear water applied.
Comparative example 2 is the use of conventional chemical control.
Comparative example 3 differs from example 1 in that only a fermentation broth of Sporotrichum roseum was applied.
Comparative example 4 is different from example 1 in that only ginkgo biloba leaf extract is administered.
comparative example 5 differs from example 1 in that only the ginkgo biloba testa extract is administered.
Comparative example 6 differs from example 1 in that only the fermentation broth of Sporotrichum roseum and the extract of Ginkgo biloba leaf are applied.
comparative example 7 differs from example 1 in that only the fermentation broth of Sporotrichum roseum and the extract of the testa Ginkgo were administered.
comparative example 8 differs from example 1 in that only ginkgo biloba leaf extract and ginkgo biloba testa extract are administered.
Lettuce plants were grown by the conventional method, and the agents of the above 6 examples and 8 comparative examples were applied, respectively, and the incidence of sclerotinia and disease index were measured at the rosette stage and the succulent stem formation stage of lettuce, respectively, and the results were recorded as in table 1 below. After emergence for 15 days, the vegetative characteristics of the lettuce seedlings were measured using a random sampling survey and the results are reported in table 2 below.
TABLE 1 Sclerotinia sclerotiorum disease onset at different times and with different treatments of lettuce
As can be seen from the data in Table 1, the incidence of sclerotinia rot of lettuce in examples 1 to 6 using the biocontrol agent of the present invention was significantly reduced, the average relative control effect in rosette stage was 85.15%, and the average relative control effect in rosette stage was increased by 22.41% as compared to comparative example 2, as compared to comparative example 1 using only clear water and comparative example 2 using a conventional chemical pesticide; the succulent stem forming period is the high incidence period of the lettuce sclerotinia sclerotiorum, the average relative prevention effect of the period examples 1-6 reaches 88.40%, and is improved by 29.67% compared with the comparative example 2, which shows that the biocontrol agent has long efficacy and better prevention and treatment effect in multiple growth periods of the lettuce.
Comparative examples 3-5 were each applied with one specific active ingredient in the biocontrol formulation, and comparative examples 6-8 were each applied with any two active combinations in the biocontrol formulation. Compared with the example 1, the result shows that the effect of preventing and treating the lettuce sclerotinia sclerotiorum is poorer by applying a specific component alone or two components, wherein the effect of the comparative example 4 of applying the ginkgo biloba extract alone and the effect of the comparative example 6 of applying the fermentation liquid of the spirillum roseum and the ginkgo biloba extract in combination are better, but the difference is larger compared with the example 1.
TABLE 2 botanical traits of lettuce seedling plants under different treatments
As can be seen from the data in Table 2, examples 1 to 6, to which the biocontrol agent of the present invention was applied, had a certain promoting effect on the growth and development of lettuce seedlings, compared to comparative example 1, to which only clear water was applied, and the plant height increased by 18.15%, the total weight increased by 32.20%, the wet weight of the above-ground part increased by 48.27%, the wet weight of the underground part increased by 30.11%, the dry weight of the above-ground part increased by 27.78%, and the dry weight of the underground part increased by 55.56%, compared to the blank control. As can be seen from the data of examples 1-6, the growth promoting effect of the lettuce seedlings is better when the biocontrol agent is used for watering the seedbed after sowing, and the growth promoting effect of leaf surface spraying on the lettuce seedlings is not obvious after the lettuce seedlings grow 5-6 leaves.
Comparative examples 3-5 were each applied with one specific active ingredient in the biocontrol formulation, and comparative examples 6-8 were each applied with any two active combinations in the biocontrol formulation. Compared with the example 1, the result shows that the effect of promoting the growth and development of lettuce seedlings by applying a specific component alone or two components in the specific component in a compounding way is poor, wherein the effect of the comparative example 3 of applying the fermentation liquor of the spirillum roseum alone and the effect of the comparative example 7 of applying the fermentation liquor of the spirillum roseum in a compounding way with the ginkgo episperm extract are better, but the difference is larger compared with the example 1.
In conclusion, the invention adopts the spirospora rosea fermentation liquor, the ginkgo leaf extract and the ginkgo episperm extract as active ingredients to be compounded according to a certain proportion, compared with a single ingredient, the compound preparation can obviously improve the control effect, can promote the growth of lettuce seedlings, can obviously inhibit the lettuce sclerotinia rot, can avoid the technical problems of easy generation of drug resistance and environmental harm in chemical control, and has a certain application prospect.
Claims (10)
1. the biocontrol agent for preventing and treating lettuce sclerotinia is characterized by comprising the following components in parts by weight: 15-35 parts of endophytic fungus spirillum roseum fermentation liquor of ginkgo, 10-20 parts of ginkgo leaf extract and 15-25 parts of ginkgo episperm extract; and (3) mixing fermentation liquor, a ginkgo leaf extract and a ginkgo episperm extract which are prepared by carrying out solid culture and liquid culture on the spirosporium roseum according to the parts by weight to obtain the biocontrol preparation.
2. The biocontrol formulation for lettuce sclerotinia as claimed in claim 1, wherein said biocontrol formulation comprises the following ingredients in parts by weight: 25 parts of endophytic fungus pinopillaria rosea fermentation liquor, 15 parts of ginkgo leaf extract and 20 parts of ginkgo episperm extract.
3. The method for preparing the biocontrol agent for controlling lettuce sclerotinia as claimed in claim 1 or 2, characterized by comprising the following steps:
(1) Inoculating activated and cultured spirillum roseum to the center of a PDA flat plate, placing the PDA flat plate in a constant-temperature incubator at 28 ℃ for culturing for 2-3d, then inoculating a purified cake of spirillum roseum to a PDB liquid culture medium, and culturing for 2-3d on a constant-temperature shaking table to obtain the spirillum roseum fermentation liquor;
(2) Drying folium Ginkgo, pulverizing, sieving with 40-60 mesh sieve, placing into an extraction tank, simultaneously adding 60-80% ethanol, extracting for 2-3 times (each time for 60-70 min), and mixing extractive solutions; distilling the extractive solution under reduced pressure to 1/5-1/10 of the original volume to obtain concentrated solution, adding purified water with the same volume into the concentrated solution, precipitating for 12-15 hr, and collecting supernatant; drying the supernatant under reduced pressure, eluting with ethanol containing acetic acid, collecting eluate, distilling the eluate under reduced pressure to obtain fluid extract, and drying the fluid extract under reduced pressure to obtain folium Ginkgo extract;
(3) decocting the ginkgo biloba sarcotesta with hot water at 90-99 ℃ for 1-2h each time for 2-3 times, wherein the material-liquid ratio is 1:6-12(W/V), filtering the decoctions, combining the filtrates, concentrating under reduced pressure, transferring the concentrated solution to a container, placing the container on a base of a magnetic stirrer, slowly dripping ethanol into the concentrated solution to make the final concentration of the ethanol reach 60-80% (V/V), stirring by starting the magnetic stirrer while adding the ethanol, standing, washing the precipitate with 80-90% ethanol for 3-5 times, and vacuum drying to obtain the ginkgo biloba sarcotesta extract;
(4) And uniformly mixing the fusarium oxysporum fermentation liquid, the ginkgo leaf extract and the ginkgo episperm extract according to parts by weight to obtain the biocontrol preparation.
4. the preparation method of the biocontrol agent for preventing and treating lettuce sclerotinia as claimed in claim 3, wherein the temperature of the constant temperature shaking culture in the step (1) is 28-30 ℃, and the rotating speed is 200-220 r/min.
5. The method for preparing a biocontrol agent for controlling lettuce sclerotinia as claimed in claim 3, characterized in that, the thallus concentration of the SPIRULINA rosea in the SPIRULINA rosea fermentation liquor obtained in the step (1) is more than or equal to 1 x 107cfu/ml。
6. The preparation method of the biocontrol agent for preventing and treating lettuce sclerotinia as claimed in claim 3, wherein the ethanol dosage in the extraction tank of the step (2) is 4-8 times of the weight of the ginkgo leaves.
7. The method for preparing a biocontrol agent for controlling lettuce sclerotinia as claimed in claim 3, wherein said ethanol containing acetic acid in step (2) is composed of ethanol with 80-90% by volume concentration and 0.1-0.5% by volume percentage of acetic acid.
8. The preparation method of the biocontrol agent for preventing and treating lettuce sclerotinia as claimed in claim 3, wherein the stirring temperature of the magnetic stirrer in the step (3) is 20-30 ℃ and the rotating speed is 1400-1800 r/min.
9. the use method of the biocontrol agent for controlling lettuce sclerotinia as claimed in claim 1 or 2, is characterized in that the use method specifically comprises the following steps: after the lettuce seeds are sowed, the biocontrol agent is taken according to 10-20ml per hundred seeds, and is diluted by 10-20 times of sterile water to be poured into a seedbed.
10. the use method of the biocontrol agent for controlling lettuce sclerotinia as claimed in claim 1 or 2, is characterized in that the use method specifically comprises the following steps: after 5-6 leaves grow on the lettuce seedlings, the biocontrol agent is diluted by 20-40 times by sterile water and is uniformly sprayed on the surfaces of the leaves of the lettuce seedling plants by a sprayer.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910652482.1A CN110558336A (en) | 2019-07-19 | 2019-07-19 | Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910652482.1A CN110558336A (en) | 2019-07-19 | 2019-07-19 | Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110558336A true CN110558336A (en) | 2019-12-13 |
Family
ID=68773013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910652482.1A Pending CN110558336A (en) | 2019-07-19 | 2019-07-19 | Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110558336A (en) |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1802925A (en) * | 2005-12-30 | 2006-07-19 | 中国计量学院 | Use of gingko endogenous fungus metabolite in preventing and treating plant fungus disease |
| CN104611243A (en) * | 2015-03-06 | 2015-05-13 | 中国农业科学院植物保护研究所 | Engineering strain-clonostachys rosea for perilipin gene transfer and application of engineering strain |
| CN105705629A (en) * | 2013-09-11 | 2016-06-22 | 蜂传播媒介技术有限公司 | Isolated strain of clonostachys rosea for use as a biological control agent |
| CN106109511A (en) * | 2016-07-19 | 2016-11-16 | 中南民族大学 | A kind of Folium Ginkgo extract and its preparation method and application |
| CN106305792A (en) * | 2016-05-19 | 2017-01-11 | 北京启高生物科技有限公司 | Clonostachys rosea-bacillus subtilis composite wettable powder |
| CN106359456A (en) * | 2016-08-27 | 2017-02-01 | 江西天人生态股份有限公司 | Botanical pesticide containing ginkgol biloba extract and preparation method thereof |
| CN106389484A (en) * | 2016-10-21 | 2017-02-15 | 扬州大学 | Methods for preparing water-soluble ginkgo exopleura extract and preparation thereof |
| CN109762743A (en) * | 2019-01-30 | 2019-05-17 | 河南省农业科学院植物保护研究所 | Gliocladium roseum, its solid fermentation microbial inoculum and its application |
-
2019
- 2019-07-19 CN CN201910652482.1A patent/CN110558336A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1802925A (en) * | 2005-12-30 | 2006-07-19 | 中国计量学院 | Use of gingko endogenous fungus metabolite in preventing and treating plant fungus disease |
| CN105705629A (en) * | 2013-09-11 | 2016-06-22 | 蜂传播媒介技术有限公司 | Isolated strain of clonostachys rosea for use as a biological control agent |
| CN104611243A (en) * | 2015-03-06 | 2015-05-13 | 中国农业科学院植物保护研究所 | Engineering strain-clonostachys rosea for perilipin gene transfer and application of engineering strain |
| CN106305792A (en) * | 2016-05-19 | 2017-01-11 | 北京启高生物科技有限公司 | Clonostachys rosea-bacillus subtilis composite wettable powder |
| CN106109511A (en) * | 2016-07-19 | 2016-11-16 | 中南民族大学 | A kind of Folium Ginkgo extract and its preparation method and application |
| CN106359456A (en) * | 2016-08-27 | 2017-02-01 | 江西天人生态股份有限公司 | Botanical pesticide containing ginkgol biloba extract and preparation method thereof |
| CN106389484A (en) * | 2016-10-21 | 2017-02-15 | 扬州大学 | Methods for preparing water-soluble ginkgo exopleura extract and preparation thereof |
| CN109762743A (en) * | 2019-01-30 | 2019-05-17 | 河南省农业科学院植物保护研究所 | Gliocladium roseum, its solid fermentation microbial inoculum and its application |
Non-Patent Citations (4)
| Title |
|---|
| M.A. RODRI´GUEZ 等，: ""Clonostachys rosea BAFC3874 as a Sclerotinia sclerotiorum antagonist: mechanisms involved and potential as a biocontrol agent"，", 《JOURNAL OF APPLIED MICROBIOLOGY》 * |
| 丁国强 等，: "《蔬菜病虫害测报实用操作手册》", 31 March 2018, 上海科学技术出版社 * |
| 于平儒 等，: ""28种植物样品提取物离体抑菌作用测定"，", 《徐州师范大学学报（自然科学版）》 * |
| 李慧明 等，: "《蔬菜病虫害预测预报调查规范》", 30 November 2006, 上海科学技术出版社 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106282067B (en) | Multifunctional agricultural complex micro organism fungicide and probiotics and application | |
| CN110074113B (en) | Application of stropharia rugoso-annulata polysaccharide, plant immunity inducer and application | |
| CN109258703A (en) | A kind of ginkgolic acid-rhamnolipid compound disinfectant and its preparation method and application | |
| CN111713510B (en) | Application of gallnut extract in preparation of plant-derived resistance inducer and resistance inducer | |
| CN113661992B (en) | Application of ligustilide and plant essential oil containing ligustilide in prevention and treatment of peanut southern blight | |
| CN113604363B (en) | Alternaria solani and application thereof | |
| CN108522176A (en) | It is a kind of promote bitter buckwheat high yield, stable yields cultural method | |
| CN107266216A (en) | A kind of plant leaf surface nutrition spray of the bent combined ferment of enzyme | |
| CN110583699B (en) | Organic preparation for preventing and treating pepper phytophthora blight and white star disease and preparation and use method thereof | |
| CN110558337A (en) | biocontrol preparation for preventing and treating rice blast and preparation method thereof | |
| CN110558336A (en) | Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof | |
| CN102176831A (en) | A process for the production of a storage stable fungicidal extract of glycyrrhiza glabra for the control of phytopathogenic fungi and other plant diseases | |
| CN101407767A (en) | Method for producing Chinese caterpillar fungus by fermentation | |
| CN103960288B (en) | The application of a kind of mixing formula preparation in control brown planthopper | |
| CN113498795A (en) | Preparation method of pure natural plant acaricide | |
| CN113767901A (en) | Application of thymol in prevention and treatment of peanut southern blight | |
| CN103960287B (en) | A kind of mixing formula preparation preventing and treating brown planthopper | |
| CN102219602B (en) | Novel biocontrol and plant vigor complexing agent and preparation method thereof | |
| CN106967622B (en) | Aspergillus flavus BP6T2 for producing paclitaxel and application thereof | |
| CN106577844B (en) | Prevent and treat capsicum epidemic disease preparation, its preparation and application | |
| CN109526974A (en) | A kind of composition and application method of efficient prevention and treatment rice blast | |
| CN112970783B (en) | Composite bait agent of artemisia sieversiana crude extract and metarhizium anisopliae for preventing and treating locusts asiaticus and application of composite bait agent | |
| CN107467073B (en) | Biological prevention and control agent containing actinomyces is used for the prevention and control and application method of Radix Notoginseng disease | |
| CN111248213B (en) | Application and method of pentacyclic triterpenoid saponin compound in aspect of improving crop yield | |
| CN117511753B (en) | Marine trichoderma strain and application thereof in prevention and treatment of tobacco mosaic virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |