CN114410511A - Bacterial powder for inhibiting tobacco black shank, preparation method and application - Google Patents
Bacterial powder for inhibiting tobacco black shank, preparation method and application Download PDFInfo
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Abstract
The invention discloses a bacterial powder for inhibiting tobacco black shank, a preparation method and application, relates to the technical field of microbial agents, and solves the technical problems that the drug resistance of pathogenic bacteria is increased year by year, pesticide residues and environmental pollution exist in the prior art when a chemical agent is adopted to prevent and control the black shank. The preparation method of the bacterial powder for inhibiting the tobacco black shank comprises the following steps: activating the strain by using the bacillus megatherium CRP-4 as the strain; culturing a bacillus megatherium CRP-4 strain to obtain a seed solution; culturing the bacillus megatherium CRP-4 seed liquid to obtain a fermentation liquid; centrifuging the fermentation liquor of the bacillus megatherium CRP-4 to obtain centrifugal thalli; and (3) carrying out spray drying on the centrifugal thallus of the bacillus megaterium CRP-4 to prepare bacillus megaterium CRP-4 powder. The prepared bacillus megatherium CRP-4 powder has obvious inhibition effect on tobacco black shank pathogenic bacteria, and lays a foundation for development of related biological preparations and field popularization and application.
Description
Technical Field
The invention relates to the technical field of microbial agents, and particularly relates to bacterial powder for inhibiting tobacco black shank, a preparation method and application.
Background
The microbial agent is an active substance which can be symbiotic, has complementary action, is artificially cultivated and can be prepared into water agent, powder or solid agent. The microbial agent contains a large amount of beneficial viable bacteria substances and a plurality of natural fermentation active substances, can be propagated in soil of a root zone to form a microbial dominant flora beneficial to the growth of crops, regulates the rhizosphere environment, assists the crops to absorb nutrition, and improves and restores the micro-ecological balance of the soil.
The diseases of rhizomes are one of the main diseases of flue-cured tobacco, wherein the diseases of black shank and bacterial wilt are the most serious and tend to spread. According to records, the bacterial wilt endangers areas, causes large-area wilting of tobacco plants if the tobacco plants are light, has large prevention and control difficulty and increases the prevention and control cost of tobacco growers; in severe disaster fields, the morbidity is as high as 30-50%, so that the whole tobacco plant is withered and dead, and the yield and the quality of tobacco leaves are seriously influenced if the disease is serious, and the enthusiasm of tobacco growers for planting tobacco is struck.
In the current tobacco planting process, the method for preventing and controlling black shank and bacterial wilt mainly comprises the following steps: the comprehensive control measures such as agricultural control measures mainly for planting disease-resistant (resistant) cured tobacco varieties, clean gardens and rounds, chemical agent control and the like. But no suitable anti (resistant) fine variety exists in the actual production; some tobacco fields have to be continuously planted due to limited cultivated land. Therefore, chemical agents are mostly used in production for controlling diseases. At present, the medicament has few varieties of alternative medicaments, the service life of a main medicament is long, the drug resistance of pathogenic bacteria is increased year by year, and the negative effects of pesticide residue, environmental pollution and the like are gradually increased. Therefore, biological control has developed into a hot field of research on control of tobacco diseases in recent years.
Disclosure of Invention
The invention aims to provide bacterial powder for inhibiting tobacco black shank, a preparation method and application, and solves the technical problems that in the prior art, the resistance of pathogenic bacteria is increased year by year, pesticide residues and environmental pollution exist when a chemical agent is adopted to prevent and control the black shank in the tobacco planting process. The various technical effects that can be produced by the preferred technical solution of the present invention are described in detail below.
In order to achieve the purpose, the invention provides the following technical scheme:
the preparation method of the bacterial powder for inhibiting the tobacco black shank comprises the following steps:
activating the strain by using the bacillus megatherium CRP-4 as the strain;
culturing the CRP-4 strain of the bacillus megaterium to obtain a CRP-4 seed solution of the bacillus megaterium;
culturing the bacillus megatherium CRP-4 seed liquid to obtain a bacillus megatherium CRP-4 fermentation liquid;
centrifuging the fermentation liquor of the bacillus megatherium CRP-4 to obtain a centrifugal thallus of the bacillus megatherium CRP-4;
and (3) carrying out spray drying on the centrifugal thallus of the bacillus megatherium CRP-4 to prepare bacillus megatherium CRP-4 bacterial powder, wherein the bacillus megatherium CRP-4 bacterial powder is bacterial powder for inhibiting the tobacco black shank.
The Bacillus megaterium (Bacillus megaterium) is CRP-4 in number, CGMCC No.18458 in preservation registration number, and is preserved in 2019, 09 and 02 days, and the preservation unit is as follows: china general microbiological culture Collection center, preservation Address: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North.
The bacillus megaterium with the number of CRP-4 is a strain separated from a greenhouse of a limited company of microbial fermentation engineering research center in Yunnan province. As can be known from the analysis result of the sequence similarity of the 16SrDNA and a phylogenetic tree, the CRP-4 has the closest genetic relationship with Bacillus megaterium, the homology is the highest, the similarity reaches 99 percent, according to the similarity and homology analysis of the phylogenetic tree, the CRP-4 and the Bacillus megaterium are positioned in the same branch, the evolutionary relationship and the distance between the CRP-4 and the Bacillus megaterium are the closest, the similarity reaches 100 percent, and the strain is identified as the Bacillus megaterium by combining morphological characteristics, culture characteristics and physiological and biochemical characteristics.
According to a preferred embodiment, the strain is Bacillus megaterium CRP-4, and the activation of the strain comprises the following steps: activating the bacillus megaterium CRP-4 stored at the temperature of minus 80 ℃ on an LB solid culture medium for 12 to 16 hours, selecting a single colony to be inoculated on the LB solid culture medium, and culturing for 1 to 2 days at the temperature of 35 ℃ to obtain the activated bacillus megaterium CRP-4.
According to a preferred embodiment, the cultivation of the strain Bacillus megaterium CRP-4 to obtain the seed solution of Bacillus megaterium CRP-4 comprises the following steps: and scraping a ring of activated bacillus megaterium CRP-4 by using an aseptic inoculating ring, inoculating the bacillus megaterium CRP-4 into 50ml of LB liquid culture medium, and culturing for 24h under the conditions that the temperature is 35 ℃ and the rotating speed is 160rpm to obtain a bacillus megaterium CRP-4 seed solution.
According to a preferred embodiment, the step of culturing the Bacillus megaterium CRP-4 seed solution to obtain the Bacillus megaterium CRP-4 fermentation solution comprises the following steps: inoculating the bacillus megaterium CRP-4 seed solution into a workshop fermentation material culture medium according to the volume ratio of the strains to the culture solution of 1: 120, and culturing for 18-24 h under the conditions that the temperature is 37 ℃ and the rotating speed is 120rpm/min to obtain the bacillus megaterium CRP-4 fermentation liquid.
According to a preferred embodiment, the step of centrifuging the fermentation liquor of the CRP-4 of the bacillus megaterium to obtain the centrifugal thallus of the CRP-4 of the bacillus megaterium comprises the following steps: pouring the fermentation liquor of the bacillus megaterium CRP-4 into a centrifuge through equipment, and preparing the centrifugal thallus of the bacillus megaterium CRP-4 under the condition that the rotation speed of the centrifuge is 4000 rpm/min.
According to a preferred embodiment, the spray drying of the centrifuged cells of the CRP-4 strain of Bacillus megaterium to obtain the CRP-4 powder of Bacillus megaterium comprises the following steps: adding a protective agent into the centrifugal thallus of the bacillus megaterium CRP-4, and transferring the centrifugal thallus of the bacillus megaterium CRP-4 added with the protective agent into spray drying equipment to obtain bacillus megaterium CRP-4 powder.
According to a preferred embodiment, the protective agent comprises the following components in parts by weight: 2 to 5 percent of diatomite, 1 to 3 percent of maltodextrin, 5 to 10 percent of calcium carbonate and the balance of water.
According to a preferred embodiment, the spray drying conditions are: the temperature of the air inlet is 120-160 ℃, the temperature of the air outlet is 40-60 ℃, and the feeding speed is 15-20 ml/min.
The bacterial powder for inhibiting the tobacco black shank is prepared by adopting the preparation method of the bacterial powder for inhibiting the tobacco black shank in any technical scheme of the invention.
The bacterial powder for inhibiting the tobacco black shank can be used in tobacco planting.
The bacterial powder for inhibiting tobacco black shank, the preparation method and the application provided by the invention at least have the following beneficial technical effects:
according to the invention, the bacillus megatherium CRP-4 is taken as a strain, and is subjected to strain activation, strain culture and centrifugal drying to prepare the bacillus megatherium CRP-4 powder, the bacillus megatherium CRP-4 powder is used in the tobacco planting process, and the bacillus megatherium CRP-4 powder has an obvious inhibition effect on tobacco black shank pathogenic bacteria under the action of the bacillus megatherium CRP-4 powder, so that a foundation is laid for developing related biological agents and popularizing and applying in fields. The bacterial powder for inhibiting the tobacco black shank, the preparation method and the application solve the technical problems that the drug resistance of pathogenic bacteria is increased year by year, pesticide residues and environmental pollution exist in the prior art when a chemical agent is adopted to prevent the black shank in the tobacco planting process. The bacillus megaterium CRP-4 also has the advantages of reliable source and easy culture.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a graph showing the inhibitory effect of Bacillus megaterium CRP-4 of the present invention on tobacco black shank;
FIG. 2 is a graph showing the effect of control experiments on the suppression of tobacco black shank.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.
The preparation method of the bacterial powder for inhibiting tobacco black shank comprises the following steps:
s1: activating the strain by using the bacillus megatherium CRP-4 as the strain;
s2: culturing the CRP-4 strain of the bacillus megaterium to obtain a CRP-4 seed solution of the bacillus megaterium;
s3: culturing the bacillus megatherium CRP-4 seed liquid to obtain a bacillus megatherium CRP-4 fermentation liquid;
s4: centrifuging the fermentation liquor of the bacillus megatherium CRP-4 to obtain a centrifugal thallus of the bacillus megatherium CRP-4;
s5: and (3) carrying out spray drying on the centrifugal thallus of the bacillus megatherium CRP-4 to prepare bacillus megatherium CRP-4 bacterial powder, wherein the bacillus megatherium CRP-4 bacterial powder is bacterial powder for inhibiting the tobacco black shank.
According to the embodiment, the bacillus megatherium CRP-4 is used as a strain, and the bacillus megatherium CRP-4 powder is prepared through strain activation, strain culture and centrifugal drying, and is used in the tobacco planting process, so that the bacillus megatherium CRP-4 powder has an obvious inhibiting effect on tobacco black shank pathogenic bacteria under the action of the bacillus megatherium CRP-4 powder, and a foundation is laid for development of related biological agents and field popularization and application. The bacterial powder for inhibiting the tobacco black shank, the preparation method and the application solve the problems that in the prior art, the resistance of pathogenic bacteria is increased year by year, pesticide residues and environmental pollution are caused by adopting chemical agents to prevent the black shank in the tobacco planting process. The bacillus megaterium CRP-4 also has the advantages of reliable source and easy culture.
According to a preferred embodiment, the strain is Bacillus megaterium CRP-4, and the activation of the strain comprises the following steps: activating the bacillus megaterium CRP-4 stored at the temperature of minus 80 ℃ on an LB solid culture medium for 12 to 16 hours, selecting a single colony to be inoculated on the LB solid culture medium, and culturing for 1 to 2 days at the temperature of 35 ℃ to obtain the activated bacillus megaterium CRP-4. Preferably, the Bacillus megaterium CRP-4 preserved at-80 deg.C can be activated on LB solid medium for 12h, 14h or 16 h. Preferably, the picked single colonies may be cultured on LB solid medium for 1 day, 1.5 days or 2 days.
According to a preferred embodiment, the cultivation of the strain Bacillus megaterium CRP-4 to obtain the seed solution of Bacillus megaterium CRP-4 comprises the following steps: and scraping a ring of activated bacillus megaterium CRP-4 by using an aseptic inoculating ring, inoculating the bacillus megaterium CRP-4 into 50ml of LB liquid culture medium, and culturing for 24h under the conditions that the temperature is 35 ℃ and the rotating speed is 160rpm to obtain a bacillus megaterium CRP-4 seed solution.
According to a preferred embodiment, the step of culturing the Bacillus megaterium CRP-4 seed solution to obtain the Bacillus megaterium CRP-4 fermentation solution comprises the following steps: inoculating the bacillus megaterium CRP-4 seed solution into a workshop fermentation material culture medium according to the volume ratio of the strains to the culture solution of 1: 120, and culturing for 18-24 h under the conditions that the temperature is 37 ℃ and the rotating speed is 120rpm/min to obtain the bacillus megaterium CRP-4 fermentation liquid. Preferably, the seed solution of the CRP-4 of the Bacillus megaterium is inoculated into a fermentation medium in a workshop, and the culture temperature is not limited to 37 ℃, and can be 36.5 ℃ or 37.5 ℃.
According to a preferred embodiment, the step of centrifuging the fermentation liquor of the CRP-4 of the bacillus megaterium to obtain the centrifugal thallus of the CRP-4 of the bacillus megaterium comprises the following steps: pouring the fermentation liquor of the bacillus megaterium CRP-4 into a centrifuge through equipment, and preparing the centrifugal thallus of the bacillus megaterium CRP-4 under the condition that the rotation speed of the centrifuge is 4000 rpm/min.
According to a preferred embodiment, the spray drying of the centrifuged cells of the CRP-4 strain of Bacillus megaterium to obtain the CRP-4 powder of Bacillus megaterium comprises the following steps: adding a protective agent into the centrifugal thallus of the bacillus megaterium CRP-4, and transferring the centrifugal thallus of the bacillus megaterium CRP-4 added with the protective agent into spray drying equipment to obtain bacillus megaterium CRP-4 powder. Preferably, the protective agent comprises the following components in parts by weight: 2 to 5 percent of diatomite, 1 to 3 percent of maltodextrin, 5 to 10 percent of calcium carbonate and the balance of water. Preferably, the air inlet temperature is 120-160 ℃, the air outlet temperature is 40-60 ℃, and the feeding speed is 15-20 ml/min. More preferably, the weight of diatomaceous earth is, for example, 2%, 3%, 4% or 5%, the weight of maltodextrin is, for example, 1%, 2% or 3%, and the weight of calcium carbonate is, for example, 5%, 7% or 10%. More preferably, the inlet temperature is, for example, 120 ℃, 140 ℃ or 160 ℃, the outlet temperature is, for example, 40 ℃, 50 ℃ or 60 ℃, and the feed rate is, for example, 15ml/min, 17ml/min or 20 ml/min.
The embodiment also provides a bacterial powder for inhibiting tobacco black shank, and the bacterial powder is prepared by adopting the preparation method of the bacterial powder for inhibiting tobacco black shank in any technical scheme in the embodiment.
The bacterial powder for inhibiting tobacco black shank can be used in tobacco planting.
The inhibitory effect of Bacillus megaterium CRP-4 on tobacco black shank is described in detail below.
Picking a loop of activated Bacillus megaterium CRP-4(Bacillus megaterium) from a plate, inoculating the loop into an LB liquid culture medium, and culturing at 160rpm and 35 ℃ for 24 hours to serve as fermentation liquor for measuring antagonistic effect;
pouring a PDA (potato 200g into blocks, boiling with water for 30min, filtering with double-layer gauze to obtain juice, adding glucose 20g, agar 15g, and purified water to 1L, naturally adjusting pH, sterilizing at 121 deg.C for 30min), placing tobacco black shank pathogenic bacteria hypha blocks in the center of the plate with a 5mm perforator, symmetrically placing 3 pieces of filter paper soaked with Bacillus megaterium CRP-4 fermentation liquid at a distance of 2.5cm from the periphery, repeating for three times with the fermentation liquid not being fermented, and culturing in an incubator at 28 deg.C for 3 days, wherein the effect of Bacillus megaterium CRP-4 on inhibiting tobacco black shank is shown in FIGS. 1 and 2. From the measurements of fig. 1, it can be seen that: the diameter of the inhibition zone of the bacillus megatherium CRP-4 to the tobacco black shank is 15 mm.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.
Claims (10)
1. A preparation method of bacterial powder for inhibiting tobacco black shank is characterized by comprising the following steps:
activating the strain by using the bacillus megatherium CRP-4 as the strain;
culturing the CRP-4 strain of the bacillus megaterium to obtain a CRP-4 seed solution of the bacillus megaterium;
culturing the bacillus megatherium CRP-4 seed liquid to obtain a bacillus megatherium CRP-4 fermentation liquid;
centrifuging the fermentation liquor of the bacillus megatherium CRP-4 to obtain a centrifugal thallus of the bacillus megatherium CRP-4;
and (3) carrying out spray drying on the centrifugal thallus of the bacillus megatherium CRP-4 to prepare bacillus megatherium CRP-4 bacterial powder, wherein the bacillus megatherium CRP-4 bacterial powder is bacterial powder for inhibiting the tobacco black shank.
2. The method for preparing bacterial powder for inhibiting tobacco black shank according to claim 1, wherein the step of activating the bacterial strain by using bacillus megatherium CRP-4 as the bacterial strain comprises the following steps: activating the bacillus megaterium CRP-4 stored at the temperature of minus 80 ℃ on an LB solid culture medium for 12 to 16 hours, selecting a single colony to be inoculated on the LB solid culture medium, and culturing for 1 to 2 days at the temperature of 35 ℃ to obtain the activated bacillus megaterium CRP-4.
3. The method for preparing bacterial powder for inhibiting tobacco black shank according to claim 1, wherein the step of culturing the bacillus megaterium CRP-4 strain to obtain the bacillus megaterium CRP-4 seed solution comprises the following steps: and scraping a ring of activated bacillus megaterium CRP-4 by using an aseptic inoculating ring, inoculating the bacillus megaterium CRP-4 into 50ml of LB liquid culture medium, and culturing for 24h under the conditions that the temperature is 35 ℃ and the rotating speed is 160rpm to obtain a bacillus megaterium CRP-4 seed solution.
4. The method for preparing bacterial powder for inhibiting tobacco black shank according to claim 1, wherein the step of culturing the bacillus megaterium CRP-4 seed solution to obtain the bacillus megaterium CRP-4 fermentation solution comprises the following steps: inoculating the bacillus megaterium CRP-4 seed solution into a workshop fermentation material culture medium according to the volume ratio of the strains to the culture solution of 1: 120, and culturing for 18-24 h under the conditions that the temperature is 37 ℃ and the rotating speed is 120rpm/min to obtain the bacillus megaterium CRP-4 fermentation liquid.
5. The method for preparing bacterial powder for inhibiting tobacco black shank according to claim 1, wherein the step of centrifuging the fermentation broth of the bacillus megaterium CRP-4 to obtain the centrifuged thallus of the bacillus megaterium CRP-4 comprises the following steps: pouring the fermentation liquor of the bacillus megaterium CRP-4 into a centrifuge through equipment, and preparing the centrifugal thallus of the bacillus megaterium CRP-4 under the condition that the rotation speed of the centrifuge is 4000 rpm/min.
6. The method for preparing bacterial powder for inhibiting tobacco black shank according to claim 1, wherein the step of spray-drying the centrifugal thalli of the bacillus megaterium CRP-4 to prepare the bacillus megaterium CRP-4 bacterial powder comprises the following steps: adding a protective agent into the centrifugal thallus of the bacillus megaterium CRP-4, and transferring the centrifugal thallus of the bacillus megaterium CRP-4 added with the protective agent into spray drying equipment to obtain bacillus megaterium CRP-4 powder.
7. The preparation method of the bacterial powder for inhibiting tobacco black shank according to claim 6, wherein the protective agent comprises the following components in parts by weight: 2 to 5 percent of diatomite, 1 to 3 percent of maltodextrin, 5 to 10 percent of calcium carbonate and the balance of water.
8. The method for preparing bacterial powder for inhibiting tobacco black shank according to claim 6, wherein the spray drying conditions are as follows: the temperature of the air inlet is 120-160 ℃, the temperature of the air outlet is 40-60 ℃, and the feeding speed is 15-20 ml/min.
9. A bacterial powder for inhibiting tobacco black shank, which is prepared by the preparation method of the bacterial powder for inhibiting tobacco black shank of any one of claims 1 to 8.
10. The use of the tobacco black shank inhibiting bacterial powder of claim 9 in tobacco planting.
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| CN202111663271.1A CN114410511A (en) | 2021-12-30 | 2021-12-30 | Bacterial powder for inhibiting tobacco black shank, preparation method and application |
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| CN115232777A (en) * | 2022-09-23 | 2022-10-25 | 云南省微生物发酵工程研究中心有限公司 | Streptomyces pseudolightyellow bacterial agent for preventing and treating tobacco black shank and application thereof |
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| CN115232777A (en) * | 2022-09-23 | 2022-10-25 | 云南省微生物发酵工程研究中心有限公司 | Streptomyces pseudolightyellow bacterial agent for preventing and treating tobacco black shank and application thereof |
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