CN106755117B - Preparation method of rice blast bacterium inhibitor - Google Patents
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- CN106755117B CN106755117B CN201611245385.3A CN201611245385A CN106755117B CN 106755117 B CN106755117 B CN 106755117B CN 201611245385 A CN201611245385 A CN 201611245385A CN 106755117 B CN106755117 B CN 106755117B
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Abstract
The invention provides a preparation method of a rice blast bacterium inhibitor, which comprises the following steps: A. cleaning fresh trifoliate fruit, grinding pulp part to homogenate, diluting with sterile water, coating on the pulp, culturing, screening bacteria with inhibitory activity to Pyricularia oryzae by using an opposing method, and selecting a strain with good antibacterial effect for later use; B. and C, inoculating the strain with good bacteriostatic effect selected in the step A or the seed liquid thereof into a PDB liquid fermentation medium, culturing at constant temperature by a shaking table to obtain a fermentation liquid, concentrating the fermentation liquid obtained in the step B under reduced pressure to paste, adding anhydrous sodium sulfate to remove water, eluting the paste with anhydrous methanol to obtain an eluent, and collecting the eluent to obtain the rice blast germ inhibitor. The magnaporthe grisea inhibitor obtained by the method has the advantages of strong activity and high purity, provides a green and environment-friendly means for preventing and controlling magnaporthe grisea, and can be directly used for experimental research.
Description
Technical Field
The invention relates to the field of extraction of active ingredients of microbial fermentation liquor, and particularly relates to a preparation method of a rice blast bacterium inhibitor.
Background
The rice blast is also called as rice fever disease, one of three major diseases of rice, the overground part of rice plants can be infected with the rice blast, and the rice blast can be divided into seedling plague, node plague, leaf plague, neck plague, grain plague and the like according to different infected parts.
The rice blast germs overwinter on the straws or rice grains in the form of conidia or mycelia. When the temperature is about 20 ℃, dormancy is released, and a large amount of conidia can be generated if proper humidity is met. Conidia are mainly transmitted by air, water and insects. When the conditions are proper, the spores are convenient for rice to germinate and invade rice cells to form diseased plants. Therefore, the rice blast can be quickly propagated, disease spots are generated, new conidia are formed, and the diseased plants are infected and diffused to the periphery to spread the rice blast.
The rice blast has the widest damage area and the greatest harm in the fungal diseases in all rice production and planting. In the epidemic years of rice blast, the yield of a severe disease area is generally reduced by 10-20%, the yield of a severe area is reduced by 40-50%, and even no grain is harvested. Grain losses are even as high as 1.57 million tons per year, calculated from a global scale, due to the presence of rice blast.
The breeding of rice resistant varieties and the application of chemical pesticides still have important effects on the prevention and the treatment of the rice blast, and simultaneously, because the speed of improving the effect of the rice blast resistant varieties is reduced, and the toxicity of chemical reagents and the drug resistance of pathogenic bacteria are caused, the effects of the breeding and the application of the chemical pesticides on the prevention and the treatment of the rice blast are weakened continuously. The research and development of biological control and biological pesticides become increasingly important under the background, the biological pesticides of plant sources and microorganism sources have remarkable control characteristics, the pressure and impact force on the environment are milder than those of chemical control, the resistance effect of the resistance of rice blast germs to the reagents can be effectively weakened due to small pressure on the environment, and the service life of the product is prolonged.
the invention discloses an active ingredient extracted by using Bacillus licheniformis and application thereof, wherein the active ingredient extraction step comprises the steps of producing a strain of Bacillus licheniformis (Bacillus licheniformis N27) with the preservation number of CGMCC No. 3585, fermenting B.licheniformis N27 by using liquid, wherein the culture medium comprises 200g of potato, 20g of glucose and 1000ml of water, the fermentation conditions comprise the temperature of 25-32 ℃ and the rotating speed of 150-250 rpm, carrying out shake culture for 2-5 days, carrying out reduced pressure concentration on the obtained fermentation liquid to obtain a crude extract, and sequentially separating the crude extract by using silica gel H, C18 and Sephadex L H-20 to obtain the active ingredient Z.
Disclosure of Invention
The invention provides a preparation method of a rice blast bacterium inhibitor, which is characterized in that a bacterial strain capable of inhibiting rice blast is screened from trifoliate fruits, and fermentation liquor of the bacterial strain is subjected to concentration, dewatering and elution to obtain the rice blast bacterium inhibitor.
A preparation method of a rice blast bacterium inhibitor comprises the following steps:
A. Screening of rice blast inhibiting strains: cleaning and disinfecting fresh trifoliate fruit, removing seeds, grinding pulp to be in a homogenate state, diluting the fruit homogenate with sterile water, coating the diluted fruit homogenate on a PDA culture medium, culturing for 1-2 days in a constant-temperature incubator at 28 ℃, selecting a single colony, screening bacteria with inhibitory activity on rice blast bacteria by using a counter-action method, and selecting a strain with good antibacterial effect for later use;
B. Preparing a strain fermentation liquor for inhibiting rice blast germs: inoculating the strain with good antibacterial effect selected in the step A into a PDB liquid fermentation medium, and performing shaking table constant-temperature culture at the temperature of 28-30 ℃ and the rpm of 120-160 for 36-48 h to obtain fermentation liquor; or inoculating the strain with good bacteriostatic effect selected in the step A to a PDB seed liquid culture medium for culture to obtain a seed liquid, inoculating the seed liquid to a PDB liquid fermentation culture medium, and performing shaking table constant-temperature culture at the temperature of 28-30 ℃ and the rpm of 120-160 for 36-48 hours to obtain a fermentation liquid;
C. Preparing an inhibitor by fermentation liquor: and C, concentrating the fermentation liquor obtained in the step B under reduced pressure to paste, adding anhydrous sodium sulfate to remove water, eluting the paste with anhydrous methanol to obtain an eluent, and collecting the eluent to obtain the rice blast fungus inhibitor.
In the step A, the cleaning and the disinfection are to treat the trifoliate fruit with 60 to 85 percent of ethanol, sterile water, 0.5 to 1.5 percent of sodium hypochlorite solution and sterile water in sequence.
In the step B, the inoculation amount of the seed liquid inoculated in the PDB liquid culture medium is 2-4%.
In the step C, when the fermentation liquor is subjected to reduced pressure concentration, the temperature of a rotary evaporator is controlled to be 42-45 ℃ and 25-35 mbar.
In the step C, the paste is eluted by adding anhydrous methanol with the volume of 1/50-1/40 of the fermentation liquid, and shaking and eluting.
In the step C, the addition amount of the anhydrous sodium sulfate is 1.5-2.5 g of anhydrous sodium sulfate per liter of fermentation liquor.
Further, the time of each shaking elution is 8-10 min.
The invention has the following beneficial effects:
1. the whole process is simple and convenient, firstly, the trifoliate fruit is treated and coated with boards to obtain the bacterial strains capable of inhibiting rice blast germs, then the fermentation liquor of the bacterial strains is concentrated into paste, anhydrous sodium sulfate is added for removing water, and then, an organic solvent anhydrous methanol is used for eluting to obtain the rice blast germ inhibitor, the rice blast inhibitor extracted from the trifoliate fruit according to the specific operation steps and the specific process parameters of the invention has strong bacteriostatic activity and high purity, and experiments prove that the bacteriostatic rate of the rice blast inhibitor to the rice blast germs is 37.79-89.89% when the concentration is 0.00625-0.1 m L/m L.
2. In the prior art, when a germ inhibitor is prepared, direct fermentation liquor is used as the inhibitor, but the inventor finds that if strain fermentation liquor which is screened from trifoliate fruits and used for inhibiting rice blast bacteria is directly used for experimental research and rice blast prevention and control, the problem of inaccurate experimental results can occur in the experimental research, so that the rice blast inhibitor is obtained by concentrating the fermentation liquor into paste, then adding anhydrous sodium sulfate and finally eluting with anhydrous methanol.
Drawings
FIG. 1 is a graph showing the inhibitory effect of a rice blast fungus inhibitor in example 3.
Detailed Description
Example 1
A preparation method of a rice blast bacterium inhibitor comprises the following steps:
A. Screening of rice blast inhibiting strains: cleaning and disinfecting fresh trifoliate fruit, removing seeds, grinding pulp to be in a homogenate state, diluting the fruit homogenate with sterile water, coating the diluted fruit homogenate on a PDA culture medium, culturing for 1-2 days in a constant-temperature incubator at 28 ℃, selecting a single colony, screening bacteria with inhibitory activity on rice blast bacteria by using a counter-action method, and selecting a strain with good antibacterial effect for later use;
B. Preparing a strain fermentation liquor for inhibiting rice blast germs: inoculating the strain with good antibacterial effect selected in the step A into a PDB liquid fermentation medium, and performing constant-temperature cultivation for 36 hours at 28-30 ℃ by using a 120rpm shaking table to obtain fermentation liquor; or inoculating the strain with good bacteriostatic effect selected in the step A to a PDB seed liquid culture medium for culture to obtain a seed liquid, inoculating the seed liquid to a PDB liquid fermentation culture medium, and performing shaking table constant-temperature culture at 28-30 ℃ and 120rpm for 36 hours to obtain a fermentation liquid;
C. Preparing an inhibitor by fermentation liquor: and C, concentrating the fermentation liquor obtained in the step B under reduced pressure to paste, adding anhydrous sodium sulfate to remove water, eluting the paste with anhydrous methanol to obtain an eluent, and collecting the eluent to obtain the rice blast fungus inhibitor.
In the step A, the cleaning and the disinfection are to treat the trifoliate fruit with 60 percent ethanol, sterile water, 0.5 percent sodium hypochlorite solution and sterile water in sequence.
In the step B, the inoculation amount of the seed liquid inoculated in the PDB liquid culture medium is 2%.
In step C, the temperature of the rotary evaporator is controlled at 42 ℃ and 25mbar when the fermentation broth is concentrated under reduced pressure.
In step C, the paste is eluted by adding anhydrous methanol with the volume of 1/50 of the fermentation liquor to shake and elute.
In step C, the anhydrous sodium sulfate was added in an amount of 1.5g anhydrous sodium sulfate per liter of fermentation broth.
Further, the time of elution is 8min for each oscillation.
Example 2
A preparation method of a rice blast bacterium inhibitor comprises the following steps:
A. Screening of rice blast inhibiting strains: cleaning and disinfecting fresh trifoliate fruit, removing seeds, grinding pulp to be in a homogenate state, diluting the fruit homogenate with sterile water, coating the diluted fruit homogenate on a PDA culture medium, culturing for 1-2 days in a constant-temperature incubator at 28 ℃, selecting a single colony, screening bacteria with inhibitory activity on rice blast bacteria by using a counter-action method, and selecting a strain with good antibacterial effect for later use;
B. Preparing a strain fermentation liquor for inhibiting rice blast germs: inoculating the strain with good bacteriostatic effect selected in the step A into a PDB liquid fermentation medium, and performing shaking table constant-temperature culture at 28-30 ℃ and 140rpm for 42 hours to obtain fermentation liquor; or inoculating the strain with good bacteriostatic effect selected in the step A to a PDB seed liquid culture medium for culture to obtain a seed liquid, inoculating the seed liquid to a PDB liquid fermentation culture medium, and performing shaking table constant-temperature culture at 28-30 ℃ and 140rpm for 48 hours to obtain a fermentation liquid;
C. Preparing an inhibitor by fermentation liquor: and C, concentrating the fermentation liquor obtained in the step B under reduced pressure to paste, adding anhydrous sodium sulfate to remove water, eluting the paste with anhydrous methanol to obtain an eluent, and collecting the eluent to obtain the rice blast fungus inhibitor.
In the step A, the cleaning and the disinfection are to treat the trifoliate fruit with 85 percent of ethanol, sterile water, 0.5 to 1.5 percent of sodium hypochlorite solution and sterile water in sequence.
In the step B, the inoculation amount of the seed liquid inoculated in the PDB liquid culture medium is 4%.
In step C, the temperature of the rotary evaporator is controlled at 45 ℃ and 35mbar when the fermentation liquor is concentrated under reduced pressure.
In step C, the paste is eluted by adding anhydrous methanol with the volume of 1/40 of the fermentation liquor to shake and elute. During elution, a small amount of anhydrous methanol is added for multiple times to elute. The eluent is sucked out by a rubber head dropper for each elution and is stored.
In step C, the anhydrous sodium sulfate was added in an amount of 2.5g per liter of fermentation broth.
Further, the time of elution is 10min for each oscillation.
Example 3
A preparation method of a rice blast bacterium inhibitor comprises the following steps:
A. Screening of rice blast inhibiting strains: cleaning and disinfecting fresh trifoliate fruit, removing seeds, grinding pulp to be in a homogenate state, diluting the fruit homogenate with sterile water, coating the diluted fruit homogenate on a PDA culture medium, culturing for 1-2 days in a constant-temperature incubator at 28 ℃, selecting a single colony, screening bacteria with inhibitory activity on rice blast bacteria by using a counter-action method, and selecting a strain with good antibacterial effect for later use;
B. Preparing a strain fermentation liquor for inhibiting rice blast germs: inoculating the strain with good antibacterial effect selected in the step A into a PDB liquid fermentation medium, and culturing for 48 hours at the constant temperature of 28-30 ℃ and a shaking table at 160rpm to obtain fermentation liquor; or inoculating the strain with good bacteriostatic effect selected in the step A to a PDB seed liquid culture medium for culture to obtain a seed liquid, inoculating the seed liquid to a PDB liquid fermentation culture medium, and performing shaking table constant-temperature culture at the temperature of 28-30 ℃ and the speed of 160rpm for 48 hours to obtain a fermentation liquid;
C. Preparing an inhibitor by fermentation liquor: and C, concentrating the fermentation liquor obtained in the step B under reduced pressure to paste, adding anhydrous sodium sulfate to remove water, eluting the paste with anhydrous methanol to obtain an eluent, and collecting the eluent to obtain the rice blast fungus inhibitor.
In the step A, the cleaning and the disinfection are to treat the trifoliate fruit with 80 percent ethanol, sterile water, 1.0 percent sodium hypochlorite solution and sterile water in sequence.
In the step B, the inoculation amount of the seed liquid inoculated in the PDB liquid culture medium is 3%.
In step C, the temperature of the rotary evaporator is controlled at 40 ℃ and 30mbar when the fermentation liquor is concentrated under reduced pressure.
In step C, the paste is eluted by adding anhydrous methanol with the volume of 1/40 of the fermentation liquor to shake and elute.
In step C, the amount of anhydrous sodium sulfate added was 2g anhydrous sodium sulfate per liter of fermentation broth.
Further, the time of elution by shaking each time is 9 min.
Bacteriostatic experiments:
(1) Preparing a culture medium: taking 400g of peeled potato, and cutting into 1cm 3decocting the left and right small pieces with distilled water until the small pieces can be broken by glass rod, filtering with 8 layers of gauze, cooling, diluting to 1000m L, preparing 2 times of solution, preparing culture medium according to Table 1, and sterilizing at 121 deg.C for 20 min.
TABLE 1
(2) after sterilization, the culture medium was cooled slightly, and divided into A, B, C three groups, each group consisting of 6 culture media, 6 culture media of group A were added with the rice blast fungus inhibitors 0m L, 0.375m L, 0.75m L0, 1.5m L1, 3m L2 and 6m L3 obtained in example 1, 6 culture media of group B were added with the rice blast fungus inhibitors 0m L4, 0.375m L5, 0.75m L6, 1.5m L7, 3m L8 and 6m L obtained in example 2, 6 culture media of group C were added with the rice blast fungus inhibitors 0m L, 0.375m L, 0.75m L, 1.5m L, 3m L and 6m L obtained in example 3, and after shaking up, plates were poured over, and 3 plates were poured over for about 20 per culture dish 20m L.
(3) After the culture medium is solidified and dried, inoculating the prepared rice blast fungus cakes with the same growth vigor in the center of each culture dish, and culturing at the constant temperature of 28 ℃ for 5-7 days. The growth of the rice blast fungus cake was observed and recorded. The effect of the magnaporthe grisea inhibitor with different concentrations on inhibiting the growth of the magnaporthe grisea cake is expressed by the inhibition rate, and the calculation formula is as follows: inhibition (%) - (1-growth diameter of the treated cake/growth diameter of the control cake) × 100%. And the growth and inhibition rates of the rice blast fungus cake of example 3 were recorded in the following table 2, and a graph of the inhibition effect was drawn based on the obtained data.
TABLE 2 example 3 growth and inhibition of Pyricularia oryzae cake concentration m L/m L, diameter (mm)
As is clear from the inhibition rate and inhibition effect graphs, the inhibition rate of rice blast germs is 37.79-89.89% when the concentration is 0.00625-0.1 m L/m L.
Claims (3)
1. A preparation method of a rice blast bacterium inhibitor is characterized by comprising the following steps: the method comprises the following steps:
A. Screening of rice blast inhibiting strains: cleaning and disinfecting fresh trifoliate fruit, removing seeds, grinding pulp to be in a homogenate state, diluting the fruit homogenate with sterile water, coating the diluted fruit homogenate on a PDA culture medium, culturing for 1-2 days in a constant-temperature incubator at 28 ℃, selecting a single colony, screening bacteria with inhibitory activity on rice blast bacteria by using a counter-action method, and selecting a strain with good antibacterial effect for later use;
B. Preparing a strain fermentation liquor for inhibiting rice blast germs: inoculating the strain with good antibacterial effect selected in the step A into a PDB liquid fermentation medium, and performing shaking table constant-temperature culture at the temperature of 28-30 ℃ and the rpm of 120-160 for 36-48 h to obtain fermentation liquor; or inoculating the strain with good bacteriostatic effect selected in the step A to a PDB seed liquid culture medium for culture to obtain a seed liquid, inoculating the seed liquid to a PDB liquid fermentation culture medium, and performing shaking table constant-temperature culture at the temperature of 28-30 ℃ and the rpm of 120-160 for 36-48 hours to obtain a fermentation liquid;
C. Preparing an inhibitor by fermentation liquor: concentrating the fermentation liquor obtained in the step B under reduced pressure to paste, adding anhydrous sodium sulfate to remove water, eluting the paste with anhydrous methanol to obtain an eluent, and collecting the eluent to obtain the rice blast fungus inhibitor; in the step C, the step of eluting the paste by using the anhydrous methanol is to add the anhydrous methanol with the volume of 1/50-1/40 of that of the fermentation liquor to carry out shaking elution; the fermentation liquor is subjected to reduced pressure concentration at 42-45 ℃ under 25-35 mbar; the addition amount of the anhydrous sodium sulfate is 1.5-2.5 g of anhydrous sodium sulfate per liter of fermentation liquor; and the time of shaking and eluting each time is 8-10 min.
2. The method for preparing a pesti-oryzae inhibitor according to claim 1, wherein: in the step A, the cleaning and the disinfection are to treat the trifoliate fruit with 60 to 85 percent of ethanol, sterile water, 0.5 to 1.5 percent of sodium hypochlorite solution and sterile water in sequence.
3. The method for preparing a pesti-oryzae inhibitor according to claim 1, wherein: in the step B, the inoculation amount of the seed liquid inoculated in the PDB liquid culture medium is 2-4%.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962623A (en) * | 2010-01-19 | 2011-02-02 | 四川农业大学 | Bacillus stain for preventing and controlling rice blast and rice sheath blight |
| CN103276044A (en) * | 2013-05-20 | 2013-09-04 | 中国农业大学 | Method for identifying rice blast biocontrol bacteria |
| CN104630111A (en) * | 2015-02-09 | 2015-05-20 | 湖南师范大学 | Bacillus amyloliquefaciens and separation method and application of active metabolites thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101962623A (en) * | 2010-01-19 | 2011-02-02 | 四川农业大学 | Bacillus stain for preventing and controlling rice blast and rice sheath blight |
| CN103276044A (en) * | 2013-05-20 | 2013-09-04 | 中国农业大学 | Method for identifying rice blast biocontrol bacteria |
| CN104630111A (en) * | 2015-02-09 | 2015-05-20 | 湖南师范大学 | Bacillus amyloliquefaciens and separation method and application of active metabolites thereof |
Non-Patent Citations (2)
| Title |
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| 水稻纹枯病及稻瘟病拮抗菌的分离与抗性成分分析;姜凯;《中农业科技辑》;20110415(第4期);全文 * |
| 水稻纹枯病及稻瘟病拮抗菌的分离与抗性成分分析;戎松杰;《农业科技辑》;20170415(第4期);全文 * |
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