CN115232777A

CN115232777A - Streptomyces pseudolightyellow bacterial agent for preventing and treating tobacco black shank and application thereof

Info

Publication number: CN115232777A
Application number: CN202211163574.1A
Authority: CN
Inventors: 杨润霞; 赵崇钧; 毛明林; 郑杨崔吉; 李�诚; 叶敏; 郭绍青
Original assignee: Microbial Fermentation Engineering Research Center Co ltd Of Yunnan Province
Current assignee: Microbial Fermentation Engineering Research Center Co ltd Of Yunnan Province
Priority date: 2022-09-23
Filing date: 2022-09-23
Publication date: 2022-10-25
Anticipated expiration: 2042-09-23
Also published as: CN115232777B; CN116804170A

Abstract

The invention discloses a streptomyces pseudolightninghamii bacterial agent for preventing and controlling tobacco black shank and application thereof, relates to the technical field of prevention and control of tobacco black shank, and solves the technical problems of pesticide residue and environmental pollution caused by the adoption of chemical pesticides for prevention and control of tobacco black shank in the prior art. The invention relates to a streptomyces pseudogriseus microbial inoculum for preventing and treating tobacco black shank, which is a streptomyces pseudogriseus fermentation broth prepared by taking streptomyces pseudogriseus as a strain and performing seed activation and at least three-stage culture. The streptomyces pseudoshallowiana microbial inoculum for preventing and controlling the tobacco black shank is used in tobacco planting, can realize effective prevention and control of the black shank, activation of soil nutrients and growth promotion of crop roots in a seedling stage, enhances the nutrient absorption of crops after effective prevention and control of diseases, and further reduces the yield loss of the crops caused by the diseases; on the other hand, the application amount of chemical pesticides can be reduced to a great extent, the environmental pollution is reduced, the crop safety is improved, and the balance of soil microbial communities is kept.

Description

Streptomyces pseudoleucotrichum microbial inoculum for preventing and treating tobacco black shank and application thereof

Technical Field

The invention relates to the technical field of tobacco black shank prevention and control, in particular to a streptomyces pseudolightgray microbial inoculum for preventing and controlling tobacco black shank and application thereof.

Background

The actinomycetes are a very abundant microbial resource, the actinomycetes capable of effectively controlling plant diseases are mainly streptomyces, spores of the actinomycetes have very strong stress resistance, and various antibiotics can be produced. The actinomycetes generally play a biological control role on pathogenic bacteria through two ways, on one hand, the actinomycetes can induce generation of bacteriostatic substances in host plant tissues or generate resistance to influence growth and reproduction of the pathogenic bacteria, and finally cause death of the pathogenic bacteria, and meanwhile, certain metabolic substances of the actinomycetes can promote plant growth, so that the optimal infection period of the pathogenic bacteria on the host plant is effectively staggered to achieve a disease prevention effect. On the other hand, the actinomycetes can reduce the pathogenicity, the infection efficiency and the inoculation thallus density of pathogenic bacteria through the antibiotic action and the competitive action, so that the population density and the pathogenicity of the pathogenic bacteria are reduced, and the prevention and treatment effect is achieved.

The comprehensive prevention and control of tobacco diseases is always an important link in tobacco production, and chemical prevention and control occupies a very important position in a tobacco disease prevention and control strategy for a long time. The large and long-term continuous use of chemical pesticides and the unreasonable use technology in tobacco production enable the tobacco root black rot germs to generate drug resistance, aggravate the environmental pollution and ecological system damage caused by pesticide use, and directly influence the tobacco quality. The biocontrol microorganism has unique advantages in the aspects of overcoming the drug resistance of plant diseases, reducing the environmental pollution and ecological damage of chemical bactericides and the residue of chemical pesticides in agricultural and sideline products, and the biological pesticide prepared by utilizing actinomycetes has the characteristics of no pollution, no residue, difficulty in causing the pest to have drug resistance, high environmental compatibility, safety to people and livestock and the like, and becomes a main body of pollution-free green pesticides and the development direction of future pesticides. Therefore, the vigorous development of biological control research on biocontrol microorganisms is an important way for agricultural sustainable development, and has important practical production significance.

Disclosure of Invention

The invention aims to provide a streptomyces pseudolight gray bacterial agent for preventing and controlling tobacco black shank and application thereof, and solves the technical problems of pesticide residue and environmental pollution caused by the adoption of chemical pesticides for preventing and controlling tobacco black shank in the prior art. 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 realize the purpose, the invention provides the following technical scheme:

the invention relates to a streptomyces pseudogriseus microbial inoculum for preventing and treating tobacco black shank, which is a streptomyces pseudogriseus fermentation broth prepared by taking streptomyces pseudogriseus as a strain and performing seed activation and at least three-stage culture.

The Streptomyces pseudograyi (Streptomyces pseudogriseolous) has the number of YWF-21-2116 and the preservation registration number of CCTCC NO: m20221096, date of deposit 2022, 07, 19 days, depository: china center for type culture Collection, collection Address: wuhan university (Wuhan city, hubei, eight-way 299 # Wuchang district).

According to a preferred embodiment, the effective viable count of Streptomyces pseudogriseus in the fermentation broth is not less than 8X 10 ⁹ cfu/ml。

The invention relates to an application of a streptomyces pseudolightninghamii microbial inoculum for preventing and treating tobacco black shank in tobacco planting.

According to a preferred embodiment, the inoculant is applied to the tobacco by one or more of root dipping, root watering, pour application, hole application.

According to a preferred embodiment, the inoculum is applied 7 days after the tobacco transplant period and 7 days after the vigorous growth period.

According to a preferred embodiment, the microbial inoculum and the seedling substrate are combined to prepare a functional seedling substrate for use.

According to a preferred embodiment, the microbial inoculum and the seedling substrate are mixed according to the weight ratio of 5-8: 1000, and the mixture is uniformly mixed for seedling of tobacco.

According to a preferred embodiment, the microbial inoculum is compatible with a fertilizer to prepare a biological fertilizer for use.

According to a preferred embodiment, the microbial inoculum and the organic fertilizer are mixed according to the weight ratio of 4-7: 1000, and the mixture is used as a base fertilizer when the tobacco is transplanted after being uniformly mixed. The streptomyces pseudolighti microbial inoculum for preventing and treating the tobacco black shank and the application thereof at least have the following beneficial technical effects:

the streptomyces pseudolighti microbial inoculum for preventing and treating the tobacco black shank is prepared by taking streptomyces pseudolighti as a strain and performing seed activation and at least three-stage culture on the streptomyces pseudolighti fermentation broth, and when the streptomyces pseudolighti fermentation broth is used for tobacco planting, the effective prevention and control of the black shank, the activation of soil nutrients and the growth promotion of crop roots can be realized in a seedling stage, the nutrient absorption of crops is enhanced after the diseases are effectively prevented and controlled, and the yield loss of the crops caused by the diseases is further reduced; on the other hand, the streptomyces pseudolightgray microbial inoculum for preventing and controlling the tobacco black shank can effectively prevent and control the tobacco soil-borne disease black shank, can reduce the application amount of chemical pesticides to a great extent, reduce environmental pollution, improve crop safety and keep the balance of soil microbial communities, namely the streptomyces pseudolightgray microbial inoculum for preventing and controlling the tobacco black shank and the application thereof solve the technical problems of pesticide residue and environmental pollution caused by the adoption of the chemical pesticides for preventing and controlling the tobacco black shank in the prior art.

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 embodiments or the prior art descriptions will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a graph showing the results of the bacteriostatic activity test of the strains selected in example 1 against tobacco phytophthora parasitica;

FIG. 2 is a graph showing the bacteriostatic results of Streptomyces pseudogriseus YWF-21-2116 fermentation broth on 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 streptomyces pseudolighti microbial inoculum for preventing and treating the tobacco black shank comprises the following steps:

step 1: the Streptomyces lividans YWF-21-2116 is used as a strain, and the strain is activated. Specifically, the Streptomyces pseudograyanous preserved by glycerol is taken out at the temperature of-80 ℃, inoculated in a liquid Gaoshi culture medium according to the inoculation amount of 3 to 5 per thousand, and cultured for 24 to 70 hours at the temperature of 28 to 30 ℃ to obtain the activated Streptomyces pseudograyanous YWF-21-2116 seed solution. More specifically, the liquid Gao's I culture medium comprises the following components in percentage by mass: 20 per mill of soluble starch, 1.0 per mill of potassium nitrate, 0.5 per mill of sodium chloride, 0.5 per mill of dipotassium hydrogen phosphate, 0.5 per mill of magnesium sulfate, 0.01 per mill of ferrous sulfate and the balance of water, and the pH is 7.5 to 8.0.

Step 2: culturing the strain of the streptomyces pseudolavelii YWF-21-2116 to obtain the primary seed solution of the streptomyces pseudolavelii YWF-21-2116. Specifically, the activated seed solution is inoculated into a primary seed culture medium according to the inoculation amount of 5 to 10 percent, and the shake culture is carried out for 24 to 70 hours under the conditions that the culture temperature is 28 to 30 ℃ and the rotation speed is 180 rpm/min, so as to obtain the Streptomyces pseudogriseus YWF-21-2116 primary seed solution. More specifically, the primary seed culture medium is a liquid Gaoshi I culture medium, and comprises the following components in parts by mass: 20 per mill of soluble starch, 1.0 per mill of potassium nitrate, 0.5 per mill of sodium chloride, 0.5 per mill of dipotassium hydrogen phosphate, 0.5 per mill of magnesium sulfate, 0.01 per mill of ferrous sulfate and the balance of water, and the pH is 7.5 to 8.0.

And step 3: culturing the first-grade seed liquid of the streptomyces pseudolavelii YWF-21-2116 to obtain the second-grade seed liquid of the streptomyces pseudolavelii YWF-21-2116. Specifically, the primary seed solution is transferred to a secondary seed culture medium in a seed tank according to the inoculation amount of 5-10%, the rotation speed is 180 rpm/min at 28-30 ℃, the ventilation volume is 1500L/h, and the tank pressure is kept at 0.04-0.06 MPa for 24-70 h, so that the Streptomyces pseudogriseus YWF-21-2116 secondary seed solution is obtained. More specifically, the secondary seed culture medium comprises the following components in percentage by mass: 20 to 30 thousandths of soybean meal, 30 to 35 thousandths of mannitol, 2 to 4 thousandths of sodium hydroxide, 1 to 2 thousandths of an antifoaming agent and the balance of water, and the pH value is 7.5 to 8.0.

And 4, step 4: culturing the second-level seed liquid of the streptomyces pseudolavendulae YWF-21-2116 to obtain the streptomyces pseudolavendulae YWF-21-2116 fermentation liquid, wherein the streptomyces pseudolavendulae YWF-21-2116 fermentation liquid is the streptomyces pseudolavendulae microbial inoculum for preventing and treating the tobacco black shank. Specifically, the secondary seed liquid is inoculated into a fermentation medium in a fermentation tank according to the inoculation amount of 5-10%, the rotation speed is 180 rpm/min at the temperature of 25-30 ℃, and the ventilation volume is 13 m ³ Culturing for 70 to 78 hours under the condition that the tank pressure is kept at 0.06 MPa, wherein the effective viable count of the streptomyces pseudolightgray YWF-21-2116 in the cultured fermentation liquid is not less than 8 multiplied by 10 ⁹ Stopping fermentation when cfu/ml, wherein the obtained fermentation liquid is the fermentation liquid of the streptomyces pseudolividans YWF-21-2116; and packaging the fermentation liquor with a microbial inoculum to form a product, namely the pseudo-light grey streptomyces microbial inoculum for preventing and treating the tobacco black shank. More specifically, the fermentation medium comprises the following components in percentage by mass: 25 to 30 per thousand of soybean meal, 30 to 40 per thousand of mannitol, 2 to 4 per thousand of sodium hydroxide, 1 to 2 per thousand of an antifoaming agent, and the balance of water, wherein the pH is 7.5 to 8.0.

The streptomyces pseudolightningensis fungicide for controlling tobacco black shank and the application thereof are described in detail below with reference to the accompanying drawings 1 and 2 of the specification and examples 1 to 4.

Example 1

This example illustrates the indoor activity assay for the Streptomyces pseudolividans YWF-21-2116 preparation of the present invention.

The preparation method of the streptomyces pseudolightningensis YWF-21-2116 preparation refers to the preparation method of the streptomyces pseudolightningensis microbial inoculum for preventing and treating tobacco black shank.

Antagonistic strains are obtained by adopting a plate confronting method. A puncher with the diameter of 5 mm is used for taking hypha blocks of pathogenic bacteria of the black shank of tobacco to inoculate in the center of a PDA culture dish, and a cross method is adopted to dip the strains by using an inoculating needle at a position 3 cm away from the center of a flat plate. Taking the treatment of only inoculating tobacco black shank pathogenic bacteria as a control, placing the flat plate in a constant temperature incubator at 28 ℃ for culturing for 7 d until the pathogenic bacteria hypha on the control flat plate grow to the edge of the culture medium, repeating the treatment for 3 times, and selecting strains with obvious inhibition zones for carrying out the determination of the antibacterial activity. The test results are shown in fig. 1.

Indoor fermentation liquor antagonistic activity detection: adopting an oxford cup method: inoculating the phytophthora parasitica to the center of a PDA culture dish, simultaneously punching a hole at a position 3 cm away from the center by using a 0.5cm puncher to remove a culture medium, sucking 80 mu L of YWF-21-2116 fermentation liquor by using a liquid transfer gun, placing the fermentation liquor in a cavity, and taking only inoculated pathogenic bacteria as a control group. And (3) placing the flat plate in a constant temperature box at 28 ℃ for culturing for 7 d, observing whether the strain fermentation liquor has an inhibiting effect on tobacco black shank pathogenic bacteria, calculating the bacteriostasis rate, and repeating each treatment for 3 times. The test results are shown in FIG. 2. Antagonistic activity tests show that the Streptomyces pseudogriseus YWF-21-2116 preparation has a strong inhibition effect on the growth of tobacco phytophthora parasitica.

Example 2

This example provides a preparation of Streptomyces pseudolividans YWF-21-2116 and a method for its direct administration as a microbial inoculum.

The preparation method of the streptomyces pseudolighti YWF-21-2116 preparation refers to the preparation method of the streptomyces pseudolighti bacterial agent for preventing and treating the tobacco black shank.

In 2021, a plot experiment is carried out in the area of dajing kylin, the experiment is carried out in a plot with serious black shank disease in 2020, the tobacco variety is K326, the drug application is carried out for 2 times respectively 7 days after the transplant period and 7 days after the vigorous growth period, every time interval is one week, the treatments are respectively 800 times of dilution of a streptomyces pseudolividans YWF-21-2116 preparation and 800 times of dilution of 93% azoxystrobin as drug controls, clear water is used as a blank control, each treatment is repeated for 3 times, plots are randomly arranged, and the management is the same as that of a common field.

Before treatment, sampling is carried out at random 10 points in a treatment area and a control area respectively, and the disease and plant rate in the field is investigated. And (4) carrying out disease investigation on the diseased plants in the disease peak period, and calculating the disease indexes before and after the medicines and the prevention and treatment effects of the morbidity. According to the GB-T23222-2008 tobacco pest and disease damage grading and investigation method.

The disease grading criteria are as follows:

level 0: the whole plant has no disease spots;

level 1: the area of the lesion spots accounts for less than 1% of the area of the whole leaf;

and 3, level: the lesion area accounts for 2 to 5 percent of the whole leaf area;

and 5, stage: the lesion area accounts for 6 to 10 percent of the whole leaf area;

and 7, stage: the lesion area accounts for 11 to 20 percent of the whole leaf area;

and 9, stage: the lesion area accounts for more than 21% of the whole leaf area.

The statistical results are shown in tables 1 and 2 below:

as can be seen from tables 1 and 2, in a plot experiment, the incidence rate of tobacco black shank can be reduced by diluting the streptomyces pseudogriseus YWF-21-2116 preparation 800 times, the control effect is equivalent to that of the commonly used azoxystrobin diluted 800 times, the control effect is superior to that of a clear water control group, the problem of pesticide residue is considered, and the application has a better application prospect compared with azoxystrobin.

Example 3

The embodiment provides a streptomycete microbial inoculum and a use method of the streptomycete microbial inoculum which is compatible with a substrate to prepare a functional seedling substrate.

The streptomycete microbial inoculum is prepared by fermenting streptomycete pseudolividans YWF-21-2116, and the effective viable count is not less than 8 multiplied by 10 ⁹ cfu/ml fermentation broth. The specific preparation method of the streptomyces microbial inoculum refers to the preparation method of the pseudo-light grey streptomyces microbial inoculum for preventing and treating the tobacco black shank.

The streptomyces inoculant can be matched with a substrate to prepare a functional seedling raising substrate for use, disease prevention and control are carried out in a seedling raising stage, and outbreak of diseases after crops are transplanted can be effectively reduced.

The specific application method is as follows: the streptomyces fungicide and the seedling substrate are mixed according to the weight ratio of 5-8: 1000, and the mixture is used for raising seedlings of crops after being uniformly mixed, and the seedlings are raised according to a conventional seedling raising mode. The crop seedlings obtained by the method have disease-resistant strains in root system matrix soil and plants during transplanting, and can effectively prevent and control tobacco black shank after transplanting. After transplanting, the streptomyces fungicide can be used for root irrigation or irrigation for 1-2 times, and the streptomyces fungicide and the seedling substrate are mixed according to the weight ratio of 6: 1000 and then used for seedling culture of crops, and the root irrigation is carried out once after 7 days of transplanting, so that the prevention and control effect can be improved by about 10%.

Example 4

The embodiment provides a streptomycete microbial inoculum and a use method of the streptomycete microbial inoculum which is compatible with an organic fertilizer to prepare a functional bio-organic fertilizer.

The streptomycete microbial inoculum is prepared by fermenting streptomycete pseudograyanomala YWF-21-2116, and the effective viable count is not less than 8 multiplied by 10 ⁹ cfu/ml fermentation broth. Specific preparation method of streptomyces microbial inoculum refers to the preparation method of the pseudo-light grey streptomyces microbial inoculum for preventing and treating tobacco black shank

The streptomyces fungicide can be compatible with an organic fertilizer to prepare a functional biological organic fertilizer for use, enters soil when a base fertilizer is applied, and is used for preventing and controlling diseases in the crop transplanting stage, so that the outbreak of the diseases after the crops are transplanted can be effectively reduced.

The specific use method is as follows: the streptomyces microbial inoculum and the organic fertilizer are mixed according to the weight ratio of 4-7: 1000, and the mixture is used as a base fertilizer after being uniformly mixed and is used when crops are transplanted.

The specific method comprises the following steps: according to the fertilizer requirement characteristics of crops, the biological organic fertilizer in the embodiment is used as a base fertilizer for transplanting the crops, the application amount is 50-200 kg/mu, conventional base fertilizer application methods such as hole application, strip application and furrow application can be adopted, transplanting and watering are carried out in time after application, spores and spores in the biological organic fertilizer are promoted to rapidly germinate in a humid environment, biological prevention and control are formed, and other measures are carried out according to conventional agricultural operations. The crop is planted according to the method, the colonization of disease-resistant strains can be formed in the soil around the root system during transplanting, and the tobacco black shank can be effectively prevented and controlled after transplanting. After transplanting, the streptomyces can be used for root irrigation or irrigation for 1-2 times, and the prevention and control effect can be improved by more than 10%. If the transplanted crop seedlings are cultivated by the seedling raising substrate in the embodiment 3, the whole-course disease control can be realized on the crops.

As can be seen from tables 2 and 4, the streptomyces fungicide and the organic fertilizer are mixed according to the weight ratio of 6: 1000 to be used as base fertilizer, and the root is irrigated once after 7 days of crop transplantation, so that the control effect is improved by about 20%.

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

1. A streptomyces pseudolavipeditus microbial inoculum for preventing and treating tobacco black shank is characterized in that streptomyces pseudolavipeditus is taken as a strain, and the strain is subjected to seed activation and at least three-stage culture to prepare streptomyces pseudolavipeditus fermentation liquor.

2. The streptomyces pseudograyi bacterial agent for preventing and treating tobacco black shank according to claim 1, wherein the effective viable count of the streptomyces pseudograyi in the fermentation liquid is not less than 8 x 10 ⁹ cfu/ml。

3. The application of the streptomyces pseudolight gray bacterial agent for preventing and treating tobacco black shank as claimed in claim 1 or 2 in tobacco planting.

4. The application of the streptomyces pseudograyiana microbial agent for controlling tobacco black shank in tobacco planting according to claim 3, wherein the microbial agent is applied to tobacco by one or more of root dipping, root irrigation, pouring and hole application.

5. The application of the streptomyces pseudolight gray bacterial agent for preventing and treating tobacco black shank in tobacco planting as claimed in claim 4, wherein the bacterial agent is applied 7 days after a tobacco transplanting period and 7 days after a vigorous growth period.

6. The application of the streptomyces pseudograyiana microbial agent for preventing and treating tobacco black shank in tobacco planting according to claim 3, wherein the microbial agent and a seedling raising matrix are combined to prepare a functional seedling raising matrix for use.

7. The application of the streptomyces pseudograyiana microbial inoculum for preventing and treating tobacco black shank in tobacco planting according to claim 6 is characterized in that the microbial inoculum and a seedling raising matrix are mixed according to the weight ratio of 5-8: 1000, and the mixture is uniformly mixed and used for raising the seedlings of tobacco.

8. The application of the streptomyces pseudograyiana microbial inoculum for controlling the tobacco black shank in tobacco planting according to claim 3, wherein the microbial inoculum is prepared into a biological fertilizer for use by being compatible with a fertilizer.

9. The application of the streptomyces pseudograyiana microbial inoculum for controlling the tobacco black shank in the tobacco planting according to claim 8 is characterized in that the microbial inoculum and an organic fertilizer are mixed according to the weight ratio of 4-7: 1000, and the mixture is used as a base fertilizer after being uniformly mixed and used in the tobacco transplanting process.