CN110384247B - Biological control method for tobacco rot disease in tobacco leaf baking process - Google Patents
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Abstract
The invention discloses a biological control method for tobacco rot during tobacco baking, and relates to the technical field of biological control of plant diseases. Before tobacco leaf baking, the biological control method sprays or soaks tobacco leaf with Bacillus mucilaginosus strain YN2011 bacterial liquid with the preservation number of CGMCC No.5724 or a microbial preparation containing the strain YN2011, and then baking is carried out. The relative prevention effect of the tobacco leaf spraying treatment on the mildew and rot diseases of the tobacco leaves in the baking process can reach more than 86.22%, and the relative prevention effect of the tobacco leaf soaking treatment on the mildew and rot diseases of the tobacco leaves in the baking process can reach more than 97.90%, so that the effect is obvious. The method has the advantages of low cost, convenient operation, no toxicity, no pollution and no residue, and is an environment-friendly method.
Description
Technical Field
The invention relates to the technical field of biological control of plant diseases, in particular to a bacillus mucilaginosus strain and a preparation thereof for controlling rotten diseases of tobacco leaves in the process of baking the tobacco leaves.
Background
The tobacco leaf rotten disease is an important infectious disease occurring in the tobacco leaf baking period (Zeng Ting Ying, etc., pathogen identification of the tobacco leaf rotten disease in the baking period, Chinese tobacco school report 2014, No. 20, No. 4: 65-68). The tobacco plants are always exposed in soil and air in the whole growth process and threatened by various moulds, and because the tobacco is rich in nutrient substances in leaves in the later growth stage, various microorganisms are easy to breed. During the tobacco leaf baking period and the storage process, once the conditions are proper, the tobacco leaves are easy to rot and deteriorate, the utilization value is basically lost, and great economic loss is brought to local tobacco growers and tobacco leaf production. Research shows that the mildew rate of tobacco leaves in the tobacco leaf baking period of the individual baking room reaches more than 50%. According to the research of Japanese ditches and constitutions, after the tobacco leaves are mildewed, the substances contained in the tobacco leaves are rapidly decomposed, the normal structure and the substances contained in the tobacco leaves are damaged, the appearance color is dull, strong mildew acid and stink are generated, the odor absorption is seriously deteriorated, when the mildewing is serious, the rotten substances are completely changed into black, dirty and stink rotten substances, the use value is lost, mycotoxin is also generated in the mildewing process, and the health of a human body is seriously influenced, such as causing human allergy, asthma and the like.
The tobacco contains various nutrient substances required by the growth of microorganisms and is widely stored in the air, tobacco stems and the surfaces of tobacco leaves of a curing barn. The tobacco leaves are polluted by the mould fungi, and the tobacco leaves can be caused to mildew under the appropriate temperature and humidity conditions due to the rich nutrition of the tobacco leaves. Yunnan province reports that the end part of the petiole of the Honghuadajinyuan tobacco leaves mildews in the baking process and is mainly caused by mildew. Zeng Ting Ying and the like identify pathogenic bacteria of rotten tobacco leaves in the curing period of Fujian province, and the rotten tobacco leaves in the curing period are confirmed to be infectious diseases caused by Rhizopus oryzae (Rhizopus oryzae Went et Geerligs) for the first time. The tobacco leaves are mildewed by a plurality of factors, and the most main physical and chemical factors are temperature, air relative humidity, illumination and the like except that the ambient air, baking facilities and the surfaces of the tobacco leaves contain a large amount of mildew. Researches show that the optimum growth temperature of rhizopus oryzae which is the pathogenic bacterium of rotten disease in the baking process is 32-40 ℃, the relative humidity is 75% -85%, the temperature is consistent with the temperature of tobacco leaves in the yellowing period of baking, and the disease is rapidly spread and spread by utilizing the growth and infection of the rhizopus oryzae.
Currently, few reports are made about tobacco leaf mildew prevention and control technologies during curing, and most tobacco leaf mildew prevention and control technologies are concentrated in the tobacco leaf storage period. The currently reported mildew preventive for tobacco production mainly comprises sodium benzoate, benzoic acid and sorbic acid, but the mildew preventive can only inhibit the growth of part of moulds and has a small bacteriostatic effect. The Wangyongwan and the like effectively prevent and treat the mildew and rot disease of the tobacco leaves during the baking period by using the chlorine dioxide and the trichloroisocyanuric acid, but the content of chlorine ions in the tobacco leaves is often overproof by using the method to prevent and treat the mildew and rot disease during the baking process. Currently, there is a lack of good biocontrol agents or products to prevent tobacco leaf mildew during curing.
Disclosure of Invention
In order to solve the technical problem, the invention provides a biological control method for tobacco rot during tobacco baking, which comprises the following steps: the effective viable count before tobacco leaf baking is 1 × 108Spraying or soaking tobacco leaves with 0-100 times of cfu/ml Bacillus mucilaginosus strain YN2011, weaving the tobacco leaves on bamboo poles according to the conventional method, airing the surface moisture of the tobacco leaves, and then putting the tobacco leaves into a baking room according to the conventional method for baking, wherein the Bacillus mucilaginosus strain YN2011 has the preservation number of: CGMCC No. 5724.
The invention provides another biological control method for controlling the rotten disease of the tobacco leaves in the tobacco leaf baking process, which comprises the following steps: before tobacco leaf baking, spraying or soaking tobacco leaves with a microbial preparation containing a Bacillus mucilaginosus (Bacillus mucilaginosus) strain YN2011, then weaving the tobacco leaves on a bamboo pole according to a conventional method, airing the moisture on the surfaces of the tobacco leaves, and then putting the tobacco leaves into a baking room according to the conventional method for baking, wherein the Bacillus mucilaginosus (Bacillus mucilaginosus) strain YN2011 is deposited according to a deposition number: CGMCC No. 5724.
The microbial preparation is fermentation liquor of a Bacillus mucilaginosus (Bacillus mucilaginosus) strain YN2011, and the effective viable count of the Bacillus mucilaginosus (Bacillus mucilaginosus) strain YN2011 in the fermentation liquor is 1 multiplied by 106More than cfu/ml.
The fermentation liquor is prepared by the following method:
(1) inoculating 1-2 rings of a Bacillus mucilaginosus strain YN2011 stored at the temperature of-80 ℃ into a seed culture medium, culturing for 24 hours at the temperature of 32 ℃ to obtain an activated Bacillus mucilaginosus strain YN2011, inoculating the activated Bacillus mucilaginosus strain YN2011 into the seed culture medium according to 1 percent of inoculation amount, and performing shake culture for 12 hours at the culture temperature of 32 ℃ and the rotating speed of 160rpm to obtain a first-level seed solution, wherein the seed culture medium is: 5-10 g/L of sucrose, 0.4-1 g/L of yeast extract, 1-2.5 g/L of ammonium sulfate, 1-2 g/L of calcium carbonate, 0.5-1 g/L of magnesium sulfate heptahydrate, 1-3 g/L of dipotassium hydrogen phosphate and 7.0-7.5 of pH;
(2) transferring the primary seed liquid to a secondary seed culture medium in a seed tank according to the inoculation amount of 1-3%, and culturing for 12h at the temperature of 30-34 ℃, the rotating speed of 100rpm, the ventilation volume of 1500L/h and the tank pressure of 0.04-0.06 MPa to obtain a secondary seed liquid; the secondary seed culture medium is the same as the seed culture medium in the step (1);
(3) inoculating the secondary seed liquid into a fermentation medium in a fermentation tank according to the inoculation amount of 1-3%, culturing for 46-48 h at 35-38 ℃, the rotating speed of 100rpm, the ventilation volume of 1500L/h and the tank pressure of 0.04-0.06 MPa until the effective viable count of a Bacillus mucilaginosus strain YN2011 in fermentation liquor is not less than 1 x 106cfu/ml, and the fermentation medium is the same as the seed medium in step (1).
In the biological control method, when the tobacco leaves are soaked, the whole leaves are soaked for more than 10 minutes.
The biological control method comprises spraying tobacco leaf at the end of leaf stalk, leaf back and leaf surface until the leaf drops.
The biological control method comprises the step of placing tobacco leaves woven on bamboo poles into a common airflow ascending type 3-4-layer intensive curing barn for curing.
Compared with the prior art, the invention has the following beneficial effects:
1. the biological control method provided by the invention can ensure that the bacillus mucilaginosus strain YN2011 colonizes in tobacco, thereby achieving the purposes of disease prevention and disease resistance. The method comprises the steps of soaking or spraying the tobacco leaves to be baked with Bacillus mucilaginosus strain YN2011 bacterial liquid or a microbial preparation thereof to control the mildew and rot disease of the tobacco leaves, spraying the tobacco leaves, wherein the relative prevention effect of the mildew and rot disease of the tobacco leaves in the baking process can reach more than 86.22%, and the relative prevention effect of the mildew and rot disease of the tobacco leaves in the tobacco leaf baking process can reach more than 97.90%.
2. The method is an environment-friendly biological control method, and has the advantages of low production cost, convenient operation, no toxicity, no pollution, no residue and the like.
Detailed Description
The present invention is further illustrated by the following examples, which are not specifically described as conventional methods.
Example 1 isolation, purification, identification and preservation of Bacillus mucilaginosus Strain YN2011
1 separation and purification of Bacillus mucilaginosus strain YN2011
1.1 materials
Collected from field soil where tobacco was planted.
1.2 preparation of the culture Medium
The silicate bacteria solid culture medium comprises: 5.0g/L of sucrose, 2.0g/L of sodium phosphate, 0.5g/L of magnesium sulfate heptahydrate, 0.1g/L of calcium carbonate, 5.0mg/L of ferric chloride hexahydrate, 15g/L of agar powder and pH of 7.0. Melting the above silicate bacteria solid culture medium, pouring into sterilized culture dish, cooling, and making into plate for separating and preserving bacteria.
1.3 isolated culture
Under aseptic condition, weighing 10g of field soil sample collected from tobacco planting, dissolving in 90mL of sterile water, sufficiently oscillating, and diluting to l0-2、10-3、l0-4Multiple soil suspensions, then 100. mu.L of each dilution was homogenizedSpreading on the above solid culture medium plate of silicate bacteria, and culturing at 37 deg.C for 3 d. Colonies that were colorless, transparent, semi-glassy, and elastic were picked and further purified until pure culture was obtained. Finally, the obtained pure culture strain was transferred into glycerol with a final concentration of 25% w/w and stored in a refrigerator at-80 ℃ under sealed conditions.
2 identification of Bacillus mucilaginosus strain YN2011
2.1 morphological characterisation
The strain preserved in 1.3 is cultured by the silicate bacteria solid culture medium for 36 hours at 32 ℃, and morphological observation and identification are carried out, so that the bacterial colony is in a round bulge with a neat edge, is colorless and transparent, is semi-glass-shaped, has a smooth and moist surface, is rich in elasticity, is difficult to pick up, and is sticky and easy to thread. The thallus is rod-shaped. Poor growth on beef extract peptone medium. When observed under a microscope, the thalli do not move, have terminal spores and produce abundant capsules on a nitrogen-free culture medium. Basically consistent with morphological characteristics of Bacillus mucilaginosus described in handbook of identifying common bacteria systems (edited by Dongxu bead et al, science publishers, 2001), the strain is preliminarily judged to belong to Bacillus mucilaginosus (YN 2011, short for: strain YN 2011.
2.2 physiological and Biochemical identification
The strain YN2011 is positive in gram staining, spore staining, starch hydrolysis, glucose utilization, citrate utilization, V-P reaction, nitrate reduction, gelatin liquefaction and the like.
2.3 molecular identification results
According to the 16S rDNA universal primer amplification sequence, according to a sequencing result, the 16S rDNA sequence of the strain YN2011 is compared with a NCBI database BLAST, the similarity between the sequence of the strain YN2011 and Bacillus mucilaginosus with the strain number of YNCUCC 0001(AY571332) is the highest and reaches 99%, and the strain YN2011 is identified as Bacillus mucilaginosus (Bacillus mucilaginosus) which is the strain YN2011 by combining morphological characteristics, physiological and biochemical characteristics and the 16S rDNA sequence of the strain.
3 deposit of Bacillus mucilaginosus (Bacillus mucoarginosus) Strain YN2011
The Bacillus mucilaginosus strain YN2011 is preserved in China general microbiological culture Collection center (CGMCC) in 1 month and 12 days 2012, the preservation number is CGMCC No.5724, and the classification and naming are as follows: bacillus mucilaginosus, was tested as viable on 1 month and 12 days 2012. The general microorganism center address: the western No. 1 Hospital No. 3 of Beijing, Chaoyang, China, has a zip code of 100101.
Example 2 preparation of a microbial preparation of the invention, comprising the steps of:
(1) inoculating 1-2 rings of a Bacillus mucilaginosus strain YN2011 stored at the temperature of-80 ℃ into a seed culture medium, culturing for 24 hours at the temperature of 32 ℃ to obtain an activated Bacillus mucilaginosus strain YN2011, inoculating the activated Bacillus mucilaginosus strain YN2011 into the seed culture medium according to 1 percent of inoculation amount, and performing shake culture for 12 hours at the culture temperature of 32 ℃ and the rotating speed of 160rpm to obtain a first-level seed solution; the seed culture medium comprises the following components: 8g/L of sucrose, 0.7g/L of yeast extract, 1.5g/L of ammonium sulfate, 1g/L of calcium carbonate, 0.8g/L of magnesium sulfate heptahydrate, 2g/L of dipotassium phosphate and pH 7.5.
(2) Transferring the primary seed liquid to a secondary seed culture medium in a seed tank according to the inoculation amount of 2%, and culturing for 12h at the temperature of 32 ℃, the rotating speed of 100rpm, the ventilation volume of 1500L/h and the tank pressure of 0.05MPa to obtain a secondary seed liquid; the secondary seed culture medium is the same as the seed culture medium in the step (1).
(3) Inoculating the secondary seed liquid into a fermentation medium in a fermentation tank according to the inoculation amount of 3%, culturing for 46-48 h at the temperature of 32 ℃, the rotating speed of 100rpm, the ventilation volume of 1500L/h and the tank pressure of 0.05MPa until the effective viable count of a Bacillus mucilaginosus strain YN2011 in fermentation liquor is not less than 1 x 106When cfu/ml is reached, the fermentation liquid is the fermentation liquid of the Bacillus mucilaginosus strain YN2011, and the fermentation liquid is the microbial preparation of the invention; the fermentation medium is the same as the seed medium in step (1).
Example 3 method for soaking tobacco leaves to be cured to biologically control mildew and rot of tobacco leaves in tobacco leaf curing process
1. Test time: 8 months in 2018-9 months in 2018
2. Test site: injexing flue-cured tobacco cooperative society of Xuanwei city of Qujing city, Yunnan province
3. Test materials: the tobacco variety to be tested is Yunyan 105, and mature and good tobacco leaves are picked in the same day.
4. And (3) experimental design: the test curing barn is a common airflow ascending type 4-layer intensive curing barn, the specifications of the curing barn are that the length, the width and the height are 8m, 2.7m and 4.1m, the circulating fan is a No. 7 axial flow fan, the controller is a HDS-6 intensive curing barn controller of Liaoning Haidi ascending machinery Limited company, and the tobacco loading of the curing barn is 45 kg.m-3. Layer 4 of the barn is the most severe region of the incidence of rotten disease and the test is conducted on layer 4.
5. The effective viable count content of Bacillus mucilaginosus strain YN2011 prepared in example 2 was 1.0X 10 by sterile water8Diluting cfu/mL microbial preparation stock solution (hereinafter referred to as microbial preparation) into 10 times of solution, 50 times of solution and 100 times of solution, soaking the tobacco leaves to be roasted in 3 concentrations, and directly picking the untreated tobacco leaves as a blank control. When the tobacco leaves are soaked in the microbial preparation for each treatment, the whole leaves are soaked for 10 min. Each treatment is totally 6 rods, each rod is woven with 70-80 pieces of tobacco leaves, the tobacco leaves are placed on the left and right sides of the layer 4 of the curing barn, one side is 3 rods, the treatment and the treatment are separated by two normal untreated tobacco leaves, and the curing is carried out according to the local conventional curing process. The test treatment comprises the following steps:
CK: directly picking up the tobacco leaves.
Treatment 1: microorganism preparation x 10, namely a diluent which is diluted by 10 times by sterile water and has the effective viable count content of 1.0 x 10 in Bacillus mucilaginosus strain YN20117cfu/mL。
And (3) treatment 2: microorganism preparation 50 times, i.e. diluted solution of microorganism preparation 50 times diluted with sterile water, and effective viable count content of Bacillus mucilaginosus strain YN2011 is 2.0 × 106cfu/mL。
And (3) treatment: microbial preparationX 100, namely diluting a diluent with 100 times of microbial preparation by using sterile water, wherein the effective viable count content of a Bacillus mucilaginosus (Bacillus mucoginosus) strain YN2011 is 1.0 x 106cfu/mL。
6. The investigation method comprises the following steps: 3 groups were randomly selected for each treatment and their disease status was recorded. The recorded disease conditions are graded according to the investigation method of Wang Yongwan (Wang Yongwan and the like. the infection of the rotten disease of the tobacco leaves in the baking period comes from prevention and treatment. Anhui agricultural science, 2017, 45(19)138-142), and are divided into six grades of 0, 1, 2, 3, 4 and 5. Grade 0 is no morbidity; grade 1, the mildew of petioles is less than 1cm, and the leaves do not attack diseases; grade 2, the petiole mildews are more than 1cm, and the leaves do not attack diseases; grade 3 is that the petioles are mildewed, and the mildewed area of the leaves is less than 25 percent; the 4 grade is that the petioles are mildewed, and the mildewed area of the leaves reaches 25 to 50 percent; grade 5 is the moldy petiole, and the moldy area of the leaf is more than 50%.
Calculating the formula:
disease index ∑ (number of diseased plant at each stage × disease grade value) × 100/(number of total investigated plants × highest grade value)
Relative control effect (%) (control area disease index-treatment area disease index) × 100/control area disease index
TABLE 1 EXAMPLE 3 prevention and treatment of mould rot of tobacco leaves during tobacco leaf curing by soaking tobacco leaves to be cured
Note: the English letters following the numbers in Table 1 indicate that the difference is significant at the P.ltoreq.0.05 level.
7. And (3) test results: the tobacco leaves are baked for 7 days, and the disease occurrence degree is counted (see table 1), the method of the invention uses the microbial preparation diluted by 10 times to achieve 99.22 percent of the control effect of the mildew and rot disease of the tobacco leaves in the tobacco leaf baking process, the microbial preparation diluted by 50 times achieves 99.08 percent of the control effect of the mildew and rot disease of the tobacco leaves in the tobacco leaf baking process, and the microbial preparation diluted by 100 times achieves 97.90 percent of the control effect of the mildew and rot disease of the tobacco leaves in the tobacco leaf baking process.
Example 4 method for spray-treating tobacco leaves to be cured to control mildew and rot of tobacco leaves in tobacco leaf curing process
The effective viable count content of Bacillus mucilaginosus strain YN2011 prepared in example 2 was 1.0X 108cfu/mL of microbial preparation stock solution (hereinafter referred to as microbial preparation), and the microbial preparation is diluted into 10 times of solution and 100 times of solution by using sterile water, 3 concentrations of the microbial preparation stock solution and the 100 times of solution are sprayed to treat the tobacco leaves to be roasted, and untreated tobacco leaves are directly picked as blank control. The microbial preparation for each treatment is sprayed on the tobacco leaves, including spraying of the end part of the leaf stalk, the leaf back and the leaf surfaces until the water drops on the leaves. Each treatment is totally 6 rods, each rod is woven with 70-80 pieces of tobacco leaves, the tobacco leaves are placed on the left and right sides of the 4 th layer of the curing barn, one side is 3 rods, the treatment and the treatment are separated by two normal untreated tobacco leaves, and the tobacco leaves are baked according to the local conventional baking process. The test treatment comprises the following steps:
CK: directly picking up the tobacco leaves.
Treatment 1: the effective viable count content of Bacillus mucilaginosus strain YN2011 of the microbial preparation is 1.0 multiplied by 108cfu/mL。
And (3) treatment 2: microorganism preparation x 10, namely a diluent which is diluted by 10 times by sterile water and has the effective viable count content of 1.0 x 10 in Bacillus mucilaginosus strain YN20117cfu/mL。
And (3) treatment: the microorganism preparation is multiplied by 100, namely, the effective viable count content of the Bacillus mucilaginosus strain YN2011 of the diluted liquid of which the microorganism preparation is diluted by 100 times by sterile water is 1.0 multiplied by 106cfu/mL。
Other processing methods similar to those in example 3 are not described again.
And (3) test results: the tobacco leaves are baked for 7 days, and the occurrence degree of the rotten diseases of the tobacco leaves is counted (see table 2). According to the method, the effect of preventing and treating the tobacco leaf rot disease in the tobacco leaf baking process by spraying the microbial preparation stock solution on the tobacco leaves to be baked reaches 95.08%, the effect of preventing and treating the tobacco leaf rot disease in the tobacco leaf baking process by spraying the microbial preparation 10 times solution on the tobacco leaves to be baked reaches 90.07%, and the effect of preventing and treating the tobacco leaf rot disease in the tobacco leaf baking process by spraying the microbial preparation 100 times solution on the tobacco leaves to be baked reaches 86.22%.
TABLE 2 example 4 prevention and treatment of mildew of tobacco leaves during the baking process by spraying the tobacco leaves to be baked
Note: the English letters following the numbers in Table 2 indicate that the difference is significant at the P.ltoreq.0.05 level.
Claims (10)
1. A biological control method for tobacco rot during tobacco baking is characterized in that: the effective viable count before tobacco leaf baking is 1 × 108cfu/ml of Bacillus mucilaginosus (C: (C))Bacillus mucilaginosus) Spraying or soaking tobacco leaves with 0-100 times of liquid of strain YN2011, weaving the tobacco leaves on a bamboo pole according to a conventional method, airing the surface moisture of the tobacco leaves, and then putting the tobacco leaves into a curing barn for curing according to the conventional method, wherein the bacillus mucilaginosus (Bacillus mucilaginosus) (YN 2011)Bacillus mucilaginosus) The strain YN2011 has a preservation number of: CGMCC No. 5724.
2. The biological control method according to claim 1, wherein the tobacco leaves woven on the bamboo poles are baked in a general air-flow rising type 3-4-layer bulk curing barn.
3. The biocontrol method of claim 1, wherein the entire leaf is soaked for more than 10 minutes while the tobacco leaves are soaked.
4. The biocontrol method of claim 1, wherein the tobacco leaves are sprayed, including spraying of the end of the petiole, the back of the leaf and the surface of the leaf, in an amount such that the leaves drip.
5. A biological control method for tobacco rot during tobacco baking is characterized in that: bacillus mucilaginosus (B) is used before tobacco leaf bakingBacillus mucilaginosus) Microorganism of strain YN2011Spraying or soaking tobacco leaf, weaving tobacco leaf on bamboo rod, air drying, and baking in baking room, wherein the Bacillus mucilaginosus isBacillus mucilaginosus) The strain YN2011 has a preservation number of: CGMCC No.5724, Bacillus mucilaginosus (B) in the microbial preparationBacillus mucilaginosus) The effective viable count of the strain YN2011 is 1 multiplied by 106More than cfu/ml.
6. The biocontrol method of claim 5, wherein the microbial agent is Bacillus mucilaginosus (Bacillus mucilaginosus)Bacillus mucilaginosus) Fermentation liquor of strain YN 2011.
7. The biocontrol method of claim 6, wherein: the fermentation liquor is prepared by the following method:
(1) bacillus mucilaginosus preserved at-80 ℃ (II)Bacillus mucilaginosus) Inoculating 1-2 rings of the strain YN2011 into a seed culture medium, and culturing at 32 ℃ for 24h to obtain activated bacillus mucilaginosus (a: (a))Bacillus mucilaginosus) Strain YN2011, 1% inoculation amount of activated bacillus mucilaginosus (Bacillus mucilaginosus)) (Bacillus mucilaginosus) Inoculating the strain YN2011 to a seed culture medium, and performing shake cultivation for 12h at the culture temperature of 32 ℃ and the rotation speed of 160rpm to obtain a first-stage seed solution, wherein the seed culture medium is as follows: 5-10 g/L of sucrose, 0.4-1 g/L of yeast extract, 1-2.5 g/L of ammonium sulfate, 1-2 g/L of calcium carbonate, 0.5-1 g/L of magnesium sulfate heptahydrate, 1-3 g/L of dipotassium hydrogen phosphate and 7.0-7.5 of pH;
(2) transferring the primary seed liquid to a secondary seed culture medium in a seed tank according to the inoculation amount of 1-3%, and culturing for 12h at the temperature of 30-34 ℃, the rotating speed of 100rpm, the ventilation volume of 1500L/h and the tank pressure of 0.04-0.06 MPa to obtain a secondary seed liquid; the secondary seed culture medium is the same as the seed culture medium in the step (1);
(3) inoculating the secondary seed liquid into a fermentation medium in a fermentation tank according to the inoculation amount of 1-3%, culturing for 46-48 h at 35-38 ℃, the rotating speed of 100rpm, the ventilation volume of 1500L/h and the tank pressure of 0.04-0.06 MPa until the culture is finishedBacillus mucilaginosus in fermentation broth: (Bacillus mucilaginosus) The effective viable count of the strain YN2011 is ≧ 1 × 106cfu/ml, and the fermentation medium is the same as the seed medium in step (1).
8. The biocontrol method of any of claims 5-7, wherein the bamboo is woven into a bamboo
And (3) placing the tobacco leaves on the rods into a common airflow ascending type 3-4-layer intensive curing barn for curing.
9. The biocontrol method of any one of claims 5-7, wherein the entire leaf is soaked for more than 10 minutes while soaking the tobacco.
10. The biological control method according to any one of claims 5 to 7, wherein, when spraying the tobacco leaves,
comprises spraying the end part of a leaf stalk, the leaf back and the leaf surface until the leaves drip.
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| CN110786532B (en) * | 2019-11-20 | 2021-07-23 | 云南省烟草农业科学研究院 | Baking method for reducing TSNAs of different varieties of flue-cured tobaccos by using strains |
| CN111869917A (en) * | 2020-07-31 | 2020-11-03 | 云南省烟草公司曲靖市公司 | Novel method for preventing and treating mildew rot in tobacco leaf baking process |
| CN112375700B (en) * | 2020-11-11 | 2022-10-21 | 云南省烟草农业科学研究院 | Bacillus belgii 05-1205, acquisition method and application thereof |
| CN114982789A (en) * | 2022-06-25 | 2022-09-02 | 湖北省烟草公司宜昌市公司 | Plant pesticide for preventing tobacco from mildew and application method thereof |
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