CN110106115B - Bacillus subtilis synergist and application thereof in preparation of bacillus subtilis microbial inoculum - Google Patents
Bacillus subtilis synergist and application thereof in preparation of bacillus subtilis microbial inoculum Download PDFInfo
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Abstract
The invention provides a bacillus subtilis synergist and application thereof in preparation of a bacillus subtilis microbial agent, and belongs to the technical field of microbial preparations. The bacillus subtilis synergist comprises the following components in parts by mass: 0.5-1.5 parts of xanthan gum, 1-3 parts of fluorescein sodium, 1-3 parts of fluorescent whitening agent CBS-X, 1-3 parts of dibutyl hydroxy toluene, 6-9 parts of cane sugar and 2-3 parts of potassium nitrate. Wherein, the xanthan gum, the fluorescein sodium and the fluorescent whitening agent CBS-X can play a good role in ultraviolet protection on the bacillus subtilis; the dibutyl hydroxy toluene can play a good role in resisting oxidation to the bacillus subtilis; the sucrose and the potassium nitrate can play an obvious synergistic role in the bacteriostatic activity of the bacillus subtilis. And the components have good biocompatibility with the bacillus subtilis.
Description
Technical Field
The invention belongs to the technical field of microbial preparations, and particularly relates to a bacillus subtilis synergist and application thereof in preparation of a bacillus subtilis microbial agent.
Background
Tobacco black shank and tobacco brown spot are important diseases in tobacco production, and the disease control depends on chemical control seriously, but the chemical control has a plurality of defects. Firstly, environmental pollution and pesticide residue are caused; secondly, the drug resistance is generated, the prevention and treatment effect is reduced, and the prevention and treatment cost is increased; thirdly, the medicament is greatly influenced by the environment and has unsatisfactory prevention and treatment effect. Compared with chemical control, biological control is not only environment-friendly, but also has the characteristics of ideal effect and the like, and is one of the research hotspots.
The bacillus subtilis Tpb55 has good control effect on various diseases, especially on tobacco scab, black shank and powdery mildew, is equivalent to the main medicament for production, and has the characteristics of promoting plant growth, improving tobacco leaf quality, increasing planting benefit and the like. The compound is processed into a preparation, and has better application prospect in preventing and treating tobacco diseases.
The processing of the formulation of microbial pesticides is more difficult than that of common chemical agents, especially live microbial pesticides. The important influencing factors are that the microorganisms are sensitive to external environmental factors such as temperature, humidity, illumination and the like, the storage stability of the preparation is poor, and the preparation is easily influenced by the environmental factors after being applied, so that the drug effect cannot be well exerted. In addition, biological agents typically have a certain difference in biological activity compared to chemical pesticides.
Another difficulty with microbial processing into formulations is that they are generally less compatible with various adjuvants than chemical pesticides, and some adjuvants may not be used at all.
Disclosure of Invention
In view of the problems in the background art, the invention aims to provide a bacillus subtilis synergist and an application thereof in preparing a bacillus subtilis microbial inoculum.
The invention provides a bacillus subtilis synergist which comprises the following components in parts by mass: 0.5-1.5 parts of xanthan gum, 1-3 parts of fluorescein sodium, 1-3 parts of fluorescent whitening agent CBS-X, 1-3 parts of dibutyl hydroxy toluene, 6-9 parts of cane sugar and 2-3 parts of potassium nitrate.
Preferably, the bacillus subtilis synergist further comprises the following components in parts by mass: 2-4 parts of sodium dodecyl sulfate, 1-3 parts of sodium methylene dinaphthalene sulfonate and 1-3 parts of polyoxyethylene glycerol ether.
Preferably, the bacillus subtilis synergist comprises the following components in parts by mass: 1 part of xanthan gum, 2 parts of fluorescein sodium, 2 parts of fluorescent whitening agent CBS-X, 2 parts of dibutyl hydroxy toluene, 7.5 parts of cane sugar, 2.5 parts of potassium nitrate, 3 parts of sodium dodecyl sulfate, 2 parts of methylene dinaphthalene sodium sulfonate and 2 parts of polyoxyethylene glycerol ether.
The invention provides an application of the bacillus subtilis synergist in preparation of a bacillus subtilis microbial inoculum.
Preferably, in the bacillus subtilis microbial inoculum, the mass ratio of the bacillus subtilis synergist to the bacillus subtilis raw powder is 20-30: 60-90.
Preferably, the preparation method of the bacillus subtilis raw powder comprises the following steps:
(1) inoculating the bacillus subtilis test-tube species into a shake flask of a meat soup culture medium, and carrying out shake culture to logarithmic phase to obtain activated strains;
(2) inoculating the activated strain into a seed tank according to the inoculation amount of 8-12% of the volume ratio of a seed tank culture medium, and culturing the activated strain in the seed tank to a logarithmic phase to obtain a seed solution;
(3) will be described inInoculating the seed solution into a production tank according to the inoculation amount of 8-12% of the volume ratio of the culture medium in the production tank, and culturing in the production tank until the final biomass is more than or equal to 1010CFU/g, the spore formation rate is more than or equal to 97 percent, and the fermentation is finished to obtain a culture solution;
(4) and filtering the culture solution, and then spray-drying to obtain the bacillus subtilis raw powder.
Preferably, the culture medium for seeding tank culture in the step (2) has the following formula: 8-12 g/L glucose and 2-4 g/L, MgSO peptone40.3-0.7 g/L, 1-2 g/L yeast extract and 1-2 g/L beef extract; the culture conditions were: the dissolved oxygen is 15-18%, the stirring speed is 150-250 rpm, and the fermentation temperature is 30-32 ℃.
Preferably, the formula of the culture medium for culturing the production tank in the step (3) is 15-25 g/L of corn flour and 40-60 g/L, MgSO of bean cake powder4·7H2O 0.4～0.6g/L，MnSO4·H2O8-12 g/L; the culture conditions were: the dissolved oxygen is 15-18%, the stirring speed is 150-250 rpm, and the fermentation temperature is 30-32 ℃.
The invention also provides a bacillus subtilis microbial inoculum, which comprises the following components in parts by mass: 70-80 parts of bacillus subtilis raw powder, 0.5-1.5 parts of xanthan gum, 1-3 parts of sodium fluorescein, 1-3 parts of fluorescent whitening agent CBS-X, 1-3 parts of dibutyl hydroxy toluene, 6-9 parts of sucrose, 2-3 parts of potassium nitrate, 2-4 parts of sodium dodecyl sulfate, 1-3 parts of methylene dinaphthalene sodium sulfonate and 1-3 parts of polyoxyethylene glyceryl ether.
Preferably, the bacillus subtilis preparation comprises the following components in parts by mass: 76 parts of bacillus subtilis raw powder, 1 part of xanthan gum, 2 parts of fluorescein sodium, 2 parts of fluorescent brightener CBS-X, 2 parts of dibutyl hydroxy toluene, 7.5 parts of sucrose, 2.5 parts of potassium nitrate, 3 parts of sodium dodecyl sulfate, 2 parts of methylene dinaphthalene sodium sulfonate and 2 parts of polyoxyethylene glycerol ether.
Has the advantages that: the invention provides a bacillus subtilis synergist which comprises the following components in parts by mass: 0.5-1.5 parts of xanthan gum, 1-3 parts of fluorescein sodium, 1-3 parts of fluorescent whitening agent CBS-X, 1-3 parts of dibutyl hydroxy toluene, 6-9 parts of cane sugar and 2-3 parts of potassium nitrate. Wherein, the xanthan gum, the fluorescein sodium and the fluorescent whitening agent CBS-X are mutually matched, and can play a good anti-ultraviolet protection role on the bacillus subtilis; the dibutyl hydroxy toluene can play a good role in resisting oxidation to the bacillus subtilis; the sucrose and the potassium nitrate can be respectively used as a carbon source and a nitrogen source of the bacillus subtilis, and can play an obvious synergistic effect on the bacteriostatic activity of the bacillus subtilis under the proportion. In addition, the components have good biocompatibility with the bacillus subtilis.
The invention also provides an application of the bacillus subtilis synergist in preparation of a bacillus subtilis microbial inoculum, wherein the bacillus subtilis synergist and bacillus subtilis raw powder are mixed according to a ratio of 20-30: 60-90, the prepared bacillus subtilis microbial inoculum has obviously improved storage stability, has good control effect on tobacco scab and black shank which are important fungal diseases of tobacco, and can promote the colonization of bacillus subtilis at the rhizosphere of tobacco.
Biological preservation Instructions
Bacillus subtilis Tpb55, which is preserved in China general microbiological culture collection center (CGMCC) at 29.12.2008, with the address of Datun road in the sunny region of Beijing, China academy of sciences, preservation number: CGMCC No. 2843.
Detailed Description
The invention provides a bacillus subtilis synergist which comprises the following components in parts by mass: 0.5-1.5 parts of xanthan gum, 1-3 parts of fluorescein sodium, 1-3 parts of fluorescent whitening agent CBS-X, 1-3 parts of dibutyl hydroxy toluene, 6-9 parts of cane sugar and 2-3 parts of potassium nitrate.
The bacillus subtilis synergist comprises xanthan gum, fluorescein sodium, a fluorescent whitening agent CBS-X, dibutyl hydroxy toluene, sucrose and potassium nitrate. In the invention, the xanthan gum is used in an amount of 0.5-1.5 parts by mass, preferably 1 part by mass; the using amount of the fluorescein sodium is 1-3 parts, preferably 2 parts; the using amount of the fluorescent whitening agent CBS-X is 1-3 parts, preferably 2 parts; the dosage of the dibutyl hydroxy toluene (BHT) is 1-3 parts, preferably 2 parts; the using amount of the sucrose is 6-9 parts, and preferably 7.5 parts; the amount of the potassium nitrate is 2-3 parts, preferably 2.5 parts. In the invention, the xanthan gum, the fluorescein sodium and the fluorescent whitening agent CBS-X are used as the ultraviolet protective agent of the bacillus subtilis, and can play a good role in ultraviolet protection on the bacillus subtilis; the dibutyl hydroxy toluene can play a good role in resisting oxidation to the bacillus subtilis; the sucrose and the potassium nitrate can be respectively used as a carbon source and a nitrogen source of the bacillus subtilis, and can play an obvious synergistic effect on the bacteriostatic activity of the bacillus subtilis under the proportion. In addition, the components have good biocompatibility with the bacillus subtilis.
The bacillus subtilis synergist also preferably comprises sodium dodecyl sulfate, sodium methylene dinaphthalene sulfonate (dispersant NNO) and polyoxyethylene glycerol ether (wetting agent MF). In the invention, the amount of the sodium dodecyl sulfate is preferably 2-4 parts by mass, and more preferably 3 parts by mass; the using amount of the sodium methylene dinaphthalene sulfonate is preferably 1-3 parts, and more preferably 2 parts; the amount of the polyoxyethylene glyceryl ether is preferably 1-3 parts, and more preferably 2 parts. In the invention, the actions of the sodium dodecyl sulfate, the sodium methylene dinaphthalene sulfonate and the polyoxyethylene glyceryl ether are respectively a suspension action, a dispersion action and a wetting action in sequence, and the three components have good biocompatibility with the bacillus subtilis.
The sources of the components in the bacillus subtilis synergist are not particularly limited, and the bacillus subtilis synergist can be any conventional and commercially available product in the field.
The invention provides an application of the bacillus subtilis synergist in preparation of a bacillus subtilis microbial inoculum. Preferably, the application comprises mixing the bacillus subtilis synergist with bacillus subtilis raw powder. In the invention, the mass ratio of the bacillus subtilis synergist to the bacillus subtilis raw powder is preferably 20-30: 60-90, more preferably 24: 76. the bacillus subtilis microbial inoculum prepared according to the proportion can play the ultraviolet protection, oxidation resistance, bacteriostasis and synergy effects of the synergist on bacillus subtilis to the maximum extent.
In the present invention, the preparation method of the bacillus subtilis raw powder preferably comprises the following steps:
(1) inoculating the bacillus subtilis test-tube species into a shake flask of a meat soup culture medium, and carrying out shake culture to logarithmic phase to obtain activated strains;
(2) inoculating the activated strain into a seed tank according to the inoculation amount of 8-12% of the volume ratio of a seed tank culture medium, and culturing the activated strain in the seed tank to a logarithmic phase to obtain a seed solution;
(3) inoculating the seed solution into a production tank according to the inoculation amount of 8-12% of the volume ratio of the culture medium of the production tank, and culturing in the production tank until the final biomass is more than or equal to 1010CFU/g, the spore formation rate is more than or equal to 97 percent, and the fermentation is finished to obtain a culture solution;
(4) filtering the culture solution, and then spray-drying to obtain the bacillus subtilis raw powder
In the invention, the bacillus subtilis in the step (1) is preferably bacillus subtilis Tpb55, which is preserved by China general microbiological culture Collection center with the preservation number: CGMCC No. 2843.
In the present invention, the medium formulation for seeding tank culture in step (2) is preferably: 8-12 g/L glucose and 2-4 g/L, MgSO peptone40.3-0.7 g/L, 1-2 g/L yeast extract and 1-2 g/L beef extract; more preferably: glucose 10g/L, peptone 3g/L, MgSO40.5g/L, 1.5g/L of yeast extract and 1.5g/L of beef extract. The culture conditions are preferably: the dissolved oxygen is 15-18%, the stirring speed is 150-250 rpm, and the fermentation temperature is 30-32 ℃; more preferably: the dissolved oxygen is 16-17%, the stirring speed is 200rpm, and the fermentation temperature is 31 ℃.
In the invention, the formula of the culture medium for culturing the production tank in the step (3) is preferably 15-25 g/L of corn flour and 40-60 g/L, MgSO of bean cake powder4·7H2O 0.4～0.6g/L，MnSO4·H2O8-12 g/L; more preferably: 20g/L of bean cake powder and 50g/L, MgSO of bean cake powder4·7H2O 0.5g/L，MnSO4·H2O10 g/L. The culture conditions are preferably: the dissolved oxygen is 15-18%, the stirring speed is 150-250 rpm, and the fermentation temperature is 30-32 ℃; more preferably: the dissolved oxygen is 16-17%, the stirring speed is 200rpm, and the fermentation temperature is 31 ℃.
The invention also provides a bacillus subtilis microbial inoculum, which comprises the following components in parts by mass: 70-80 parts of bacillus subtilis raw powder, 0.5-1.5 parts of xanthan gum, 1-3 parts of sodium fluorescein, 1-3 parts of fluorescent whitening agent CBS-X, 1-3 parts of dibutyl hydroxy toluene, 6-9 parts of sucrose, 2-3 parts of potassium nitrate, 2-4 parts of sodium dodecyl sulfate, 1-3 parts of methylene dinaphthalene sodium sulfonate and 1-3 parts of polyoxyethylene glyceryl ether. Preferably, the bacillus subtilis preparation comprises the following components in parts by mass: 76 parts of bacillus subtilis raw powder, 1 part of xanthan gum, 2 parts of fluorescein sodium, 2 parts of fluorescent brightener CBS-X, 2 parts of dibutyl hydroxy toluene, 7.5 parts of sucrose, 2.5 parts of potassium nitrate, 3 parts of sodium dodecyl sulfate, 2 parts of methylene dinaphthalene sodium sulfonate and 2 parts of polyoxyethylene glycerol ether.
Compared with the conventional microbial inoculum, the bacillus subtilis microbial inoculum has the advantages that the storage period stability is improved, the field colonization capacity is improved, and the disease control effect is improved.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1g of xanthan gum, 2g of sodium fluorescein, 2g of fluorescent whitening agent CBS-X, 2g of dibutylhydroxytoluene, 7.5g of sucrose and 2.5g of potassium nitrate were weighed. And then uniformly mixing the weighed raw materials, controlling the processing temperature to be about 25 ℃, crushing in a jet mill, and sieving with a 150-mesh sieve to obtain the bacillus subtilis synergist.
Example 2
Weighing 1g of xanthan gum, 2g of sodium fluorescein, 2g of fluorescent whitening agent CBS-X, 2g of dibutyl hydroxy toluene, 7.5g of sucrose, 2.5g of potassium nitrate, 3g of sodium dodecyl sulfate, 2g of methylene dinaphthalene sodium sulfonate and 2g of polyoxyethylene glycerol ether. And then uniformly mixing the weighed raw materials, controlling the processing temperature to be about 25 ℃, crushing in a jet mill, and sieving with a 150-mesh sieve to obtain the bacillus subtilis synergist.
Example 3
(1) 200 hundred million live spores/gram of bacillus subtilis Tpb55 raw powder was prepared:
inoculating Bacillus subtilis Tpb55 test-tube strains into a shake flask of a meat soup culture medium, and carrying out shake culture to logarithmic phase;
inoculating the cultured strain into a 30L seeding tank according to the inoculum size of 10 percent of the volume ratio of the seeding tank culture medium, and culturing to logarithmic phase; the culture medium formula used by the seeding tank is as follows: glucose 10g/L, peptone 3g/L, MgSO40.5g/L, 1.5g/L of yeast extract and 1.5g/L of beef extract; the culture conditions are as follows: the dissolved oxygen is 15-18%, the stirring speed is 200rpm, and the fermentation temperature is 31 ℃;
inoculating the seed liquid into a production tank for culture according to the inoculation amount of 10% of the culture medium of the production tank by volume ratio, wherein the culture medium of the production tank has the formula: corn flour 20g/L, bean cake powder 50g/L, MgSO4·7H2O 0.5g/L，MnSO4·H2O10 g/L; the culture conditions are as follows: the dissolved oxygen is 15-18%, the stirring speed is 200rpm, and the fermentation temperature is 31 ℃; culturing until the final biomass is 1010CFU/g, the spore formation rate reaches more than 97 percent, and the fermentation is finished;
fourthly, after the fermentation is finished, the culture solution is taken out of the tank, filtered and spray-dried to obtain the raw powder of the bacillus subtilis Tpb 55.
(2) Weighing 76g of the raw powder of the bacillus subtilis Tpb55 prepared in the step (1), then uniformly mixing the bacillus subtilis synergist (totally 24g) prepared in the embodiment 2 with the raw powder of the bacillus subtilis Tpb55, controlling the processing temperature to be about 25 ℃, crushing in an airflow crusher, and sieving with a 150-mesh sieve to obtain the bacillus subtilis microbial inoculum.
Example 4
(1) 200 hundred million live spores/gram of bacillus subtilis Tpb55 raw powder was prepared:
inoculating Bacillus subtilis Tpb55 test-tube strains into a shake flask of a meat soup culture medium, and carrying out shake culture to logarithmic phase;
inoculating the cultured strain into a 30L seeding tank according to the inoculum size of 10 percent of the volume ratio of the seeding tank culture medium, and culturing to logarithmic phase; the culture medium formula used by the seeding tank is as follows: glucose 10g/L, peptone 3g/L, MgSO40.5g/L, 1.5g/L of yeast extract and 1.5g/L of beef extract; culture stripA piece: the dissolved oxygen is 15-18%, the stirring speed is 200rpm, and the fermentation temperature is 31 ℃;
inoculating the seed liquid into a production tank for culture according to the inoculation amount of 10% of the culture medium of the production tank by volume ratio, wherein the culture medium of the production tank has the formula: corn flour 20g/L, bean cake powder 50g/L, MgSO4·7H2O 0.5g/L，MnSO4·H2O10 g/L; the culture conditions are as follows: the dissolved oxygen is 15-18%, the stirring speed is 200rpm, and the fermentation temperature is 31 ℃; culturing until the final biomass is 1010CFU/g, the spore formation rate reaches more than 97 percent, and the fermentation is finished;
fourthly, after the fermentation is finished, the culture solution is taken out of the tank, filtered and spray-dried to obtain the raw powder of the bacillus subtilis Tpb 55.
(2) Weighing 76g of the bacillus subtilis Tpb55 raw powder prepared in the step (1); weighing 1g of xanthan gum, 2g of sodium fluorescein, 2g of fluorescent whitening agent CBS-X, 2g of dibutyl hydroxy toluene, 7.5g of sucrose, 2.5g of potassium nitrate, 3g of sodium dodecyl sulfate, 2g of methylene dinaphthalene sodium sulfonate and 2g of polyoxyethylene glycerol ether. And then uniformly mixing all the weighed raw materials, controlling the processing temperature to be about 25 ℃, crushing in a jet mill, and sieving with a 150-mesh sieve to obtain the bacillus subtilis microbial inoculum.
Example 5
The influence of the synergist on the in vitro bacteriostatic activity of the bacillus subtilis is determined by adopting a medicine-containing culture medium method:
3mL of diluted bacillus subtilis secondary metabolite is added into 27mL of PDA culture medium cooled to 45 ℃ to prepare a drug-containing culture medium plate with the required final concentration. Then preparing 6mm diameter mycelium blocks from the edges of the bacterial colonies of the tobacco black shank pathogenic bacteria and the tobacco brown spot pathogenic bacteria cultured for 7 days, transferring the mycelium blocks to each series of drug-containing culture media with the mycelium surfaces facing downwards, and repeating the treatment for 3 times. After the treatment, the mixture is placed in a constant-temperature biochemical incubator at 28 ℃ for culture. After 5d, the colony diameter is investigated, and the bacteriostasis rate is calculated. The test results are shown in Table 1.
Table 1 shows that the bacillus subtilis has better inhibition effect on tobacco phytophthora parasitica and tobacco brown spot pathogenic bacteria, and the inhibition effect is increased along with the increase of the concentration of the bacillus subtilis. The synergist provided in example 2 (Tpb55: synergist 80:20) was added according to design, and the bacteriostatic effect of Bacillus subtilis was improved to some extent. Aiming at tobacco phytophthora parasitica, the inhibition effect of 3 concentrations of the added synergist is respectively improved by 7.18 percent, 14.35 percent and 4.22 percent compared with the inhibition effect of bacillus subtilis with the corresponding concentration of no synergist. Aiming at the tobacco alternaria alternate pathogenic bacteria, the inhibition effect of 3 concentrations of the added synergist is respectively improved by 5.48 percent, 4.11 percent and 4.43 percent compared with the inhibition effect of bacillus subtilis with the corresponding concentration of no synergist.
TABLE 1 synergistic Effect of synergists on in vitro bacteriostatic Activity of Bacillus subtilis
Example 6
(1) Effect of synergist on storage of Bacillus subtilis Tpb55 at room temperature
The synergist provided in example 2 was added to Bacillus subtilis Tpb55 room temperature inoculant, and the mixture was stored at room temperature for 360 days, with the spore content change shown in Table 2.
Table 2 shows: the synergist can obviously improve the survival rate of the bacillus subtilis Tpb55 wettable powder when the bacillus subtilis Tpb55 wettable powder is stored at room temperature. After the bacillus subtilis is stored for 360 days at room temperature, the survival rate of spores of the bacillus subtilis agent processed by the aid is 74.19%, and is improved by 9.9% compared with the method without adding the synergist.
TABLE 2 Effect of adjuvants on the survival Rate of Bacillus subtilis Tpb55 inoculum during storage (%)
(2) Influence of synergist on effect of bacillus subtilis Tpb55 on preventing and treating tobacco brown spot
The tobacco brown spot prevention and treatment test is arranged in 2017 in Qingdao city, namely black tobacco field, and the cured tobacco variety is NC 89. The transplanting period of 2017 is 5 months and 17 days at the beginning of disease onsetThe application is started. Tpb55 microbial inoculum (Tpb55: 80:20) added with the synergist provided in the embodiment 2 is continuously sprayed on 20 days in 7 months, 30 days in 7 months and 7 days in 8 months, the Tpb55 microbial inoculum 400 times liquid without the synergist is used as a contrast, the 40% dimethachlon wettable powder 500 times liquid is used as a medicament contrast, and clear water is used as a blank contrast. Cell area 66.7m2100 tobacco plants were planted per cell. Disease index was investigated at 20 days 7 and 17 days 8. The test results are shown in Table 3.
Table 3 shows that the prevention and treatment effect of the Tpb55 microbial inoculum and the synergist after being compounded is higher than that of the tobacco alternaria alternate without being added with the synergist, and the prevention and treatment effect is equivalent to that of the main medicament dimethachlon in production. When the pesticide is used for preventing and treating the brown spot in production, the dosage of 125 g per mu is preferably used, the pesticide is not used in the field or in the initial stage of the disease, and the pesticide is continuously applied for 2-3 times at intervals of 7-10 days.
TABLE 3 field control of tobacco brown spot by Tpb55 microbial inoculum (2017)
(3) Influence of synergist on tobacco black shank prevention and treatment effect of bacillus subtilis Tpb55
The test for preventing and treating the tobacco black shank is arranged to be carried out in 2017 in a black shank disease nursery at an inky experiment station of Chinese agricultural research tobacco institute, the test land is a plain field block, and the soil is eluviated brown soil. Transplanting in 6 months and 8 days, wherein the row spacing is 100cm, the plant spacing is 30cm, and the cultivation conditions of all experimental communities are consistent. And 5 g/strain of the phytophthora parasitica strain valley is artificially inoculated in 5 days after 7 months. The cultivation management is carried out according to the unified production standard of the experimental field, and accords with the agricultural practice (GAP) of local science. Applying the pesticide 3 times, dipping the roots during transplanting, and transplanting the seedlings 2 times, namely 6 months 15 days and 6 months 27 days, wherein the using method is root irrigation, the pesticide amount per mu is 50 liters, and each plant is 100 mL. Day 7, 15, survey until the control black shank reached moderate onset. The test results are shown in Table 4.
Table 4 shows that the control effect of the Tpb55 on the tobacco black shank is better than that of the single Tpb55 after the synergist provided in example 2 is added. When the plant growth regulator is used for preventing and treating black shank in production, the dosage of 125 g per mu is preferably used, the roots are soaked for 1h in transplanting, the roots are irrigated for 1 time 7-10 days after transplanting, and the roots are irrigated for one time 15-20 days after transplanting.
TABLE 4 field control of tobacco Black shank by Tpb55 bacterial agent (2017)
(4) Influence of synergist on rhizosphere colonization condition of bacillus subtilis Tpb55 microbial inoculum
The tobacco rhizosphere colonization was determined using GFP-tagged Tpb55 strain and the results are shown in table 5. Table 5 shows: the synergist provided in example 2 can significantly improve the colonization ability of the Tpb55 strain in the tobacco rhizosphere.
TABLE 5 Effect of synergists on rhizosphere colonization by Bacillus subtilis Tpb55 inoculum
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. The bacillus subtilis synergist is characterized by comprising the following components in parts by mass: 0.5-1.5 parts of xanthan gum, 1-3 parts of fluorescein sodium, 1-3 parts of fluorescent brightener CBS-X, 1-3 parts of dibutyl hydroxy toluene, 6-9 parts of cane sugar, 2-3 parts of potassium nitrate, 2-4 parts of sodium dodecyl sulfate, 1-3 parts of methylene dinaphthalene sodium sulfonate and 1-3 parts of polyoxyethylene glycerol ether.
2. The bacillus subtilis synergist according to claim 1, which is characterized by comprising the following components in parts by mass: 1 part of xanthan gum, 2 parts of fluorescein sodium, 2 parts of fluorescent whitening agent CBS-X, 2 parts of dibutyl hydroxy toluene, 7.5 parts of cane sugar, 2.5 parts of potassium nitrate, 3 parts of sodium dodecyl sulfate, 2 parts of methylene dinaphthalene sodium sulfonate and 2 parts of polyoxyethylene glycerol ether.
3. The use of a bacillus subtilis synergist according to any one of claims 1-2 in a bacillus subtilis preparation.
4. The application of claim 3, wherein in the Bacillus subtilis microbial inoculum, the mass ratio of the Bacillus subtilis synergist to the Bacillus subtilis raw powder is 20-30: 60-90.
5. The use according to claim 4, wherein the preparation method of the Bacillus subtilis raw powder comprises the following steps:
(1) inoculating the bacillus subtilis test-tube species into a shake flask of a meat soup culture medium, and carrying out shake culture to logarithmic phase to obtain activated strains;
(2) inoculating the activated strain into a seed tank according to the inoculation amount of 8-12% of the volume ratio of a seed tank culture medium, and culturing the activated strain in the seed tank to a logarithmic phase to obtain a seed solution;
(3) inoculating the seed solution into a production tank according to the inoculation amount of 8-12% of the volume ratio of the culture medium of the production tank, and culturing in the production tank until the final biomass is more than or equal to 1010CFU/g, the spore formation rate is more than or equal to 97 percent, and the fermentation is finished to obtain a culture solution;
(4) and filtering the culture solution, and then spray-drying to obtain the bacillus subtilis raw powder.
6. The use of claim 5, wherein the culture medium for seeding tank culture in step (2) is formulated as: 8-12 g/L glucose and 2-4 g/L, MgSO peptone40.3-0.7 g/L, 1-2 g/L yeast extract and 1-2 g/L beef extract; the culture conditions were: the dissolved oxygen is 15-18%, the stirring speed is 150-250 rpm, and the fermentation temperature is 30-32 ℃.
7. The use of claim 5, wherein the culture medium for culturing the production tank in the step (3) comprises 15-25 g/L of corn flour and 40-60 g/L, MgSO of bean cake flour4·7H2O 0.4～0.6g/L，MnSO4·H2O8-12 g/L; the culture conditions were: the dissolved oxygen is 15-18%, the stirring speed is 150-250 rpm, and the fermentation temperature is 30-32 ℃.
8. The bacillus subtilis preparation is characterized by comprising the following components in parts by mass: 70-80 parts of bacillus subtilis raw powder, 0.5-1.5 parts of xanthan gum, 1-3 parts of sodium fluorescein, 1-3 parts of fluorescent whitening agent CBS-X, 1-3 parts of dibutyl hydroxy toluene, 6-9 parts of sucrose, 2-3 parts of potassium nitrate, 2-4 parts of sodium dodecyl sulfate, 1-3 parts of methylene dinaphthalene sodium sulfonate and 1-3 parts of polyoxyethylene glyceryl ether.
9. The bacillus subtilis agent of claim 8, which is characterized by comprising the following components in parts by mass: 76 parts of bacillus subtilis raw powder, 1 part of xanthan gum, 2 parts of fluorescein sodium, 2 parts of fluorescent brightener CBS-X, 2 parts of dibutyl hydroxy toluene, 7.5 parts of sucrose, 2.5 parts of potassium nitrate, 3 parts of sodium dodecyl sulfate, 2 parts of methylene dinaphthalene sodium sulfonate and 2 parts of polyoxyethylene glycerol ether.
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| CN117511749B (en) * | 2023-10-31 | 2024-07-09 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Solid state fermentation method for trichoderma asperellum HG1 and bacillus subtilis Tpb55 and application thereof |
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