CN117229935A - Bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and culture method thereof - Google Patents
Bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and culture method thereof Download PDFInfo
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Abstract
A bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and a culture method thereof relate to the technical field of bacillus polymyxa culture. The method comprises the following specific steps: activating strains; culturing bacterial liquid; preparing a special culture medium: the special culture medium has nutrition supply capacity to the bacillus polymyxa T79 which exceeds that of an LB culture medium, and under the same culture condition, the content of the special culture medium is 6 times higher than that of the bacillus polymyxa T79; and (5) producing bacterial liquid or mother strain. The invention has the beneficial effects that: by adopting the culture method to culture the bacillus polymyxa T79, the bacillus polymyxa T79 can be cultured more stably, the culture efficiency is more efficient, and the bacillus polymyxa T79 can be better applied to different fields.
Description
Technical Field
The invention relates to the technical field of culture of bacillus polymyxa, in particular to bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and a culture method thereof.
Background
Paenibacillus polymyxa (Paenibacillus polymyxa) is an aerobic bacillus, facultative anaerobic, and can decompose glucose and other saccharides to produce acid, and sometimes produce gas, and has no soluble pigment on nutrient agar. Paenibacillus polymyxa is widely inhabited in natural environment, is common in soil, plant body surfaces and root and low stem parts of plants, is common plant endophyte, and is characterized in that the isolated Paenibacillus polymyxa strain in nature has certain biological activity, such as antibiotic action, bacteriolysis, induced resistance, growth promoting action and the like, and has antiviral, antitumor, mycoplasma and antifungal biological activity, so that the Paenibacillus polymyxa can well grow in hypertonic, high-acid, high-alkali, high-temperature and high-cold environments and has great ecological value, therefore, the Paenibacillus polymyxa has wide application value in the fields of industry, medicine, agriculture and the like, and field experiments of the Paenibacillus polymyxa treated plants show that the Paenibacillus polymyxa activity comprises nitrogen fixation, phosphorus in the dissolved soil, antibiotics, hormones, cytokinins, chitinase and hydrolase are generated, the expression of related genes of the induced plants is improved in soil structure. These activities may play an important role in promoting plant growth.
Bacillus polymyxa T79 (Paenibacillus polymyxa T, 79) was isolated from honeysuckle rhizosphere soil and identified as Bacillus polymyxa by 16S and gyrB methods. Through the opposition of the flat plates, the bacillus polymyxa T79 is found to have antagonistic activity on 8 pathogenic fungi such as fusarium oxysporum, alternaria alternata, phomopsis, septoria rosea, trichothecene lycopersicum, penicillium and aspergillus. The pathogenicity of bacillus polymyxa T79 is identified, and the bacillus polymyxa T79 has no pathogenicity on honeysuckle and other plants.
In the control effect test, the effectiveness test is carried out on the bacillus polymyxa T79 strain by adopting an outdoor developed honeysuckle disease strain, after the vibration culture bacterial suspension is diluted by tap water, the vibration culture bacterial suspension is sprayed on the honeysuckle powdery mildew strain by using a manual watering can, 3 strains at different positions are used as one treatment, 27 leaf powdery mildew spot diameters and 27 new branch lengths in different directions are marked, and the average value is calculated. Water control was set up for a total of 7 treatments. The change condition of powdery mildew spots is recorded by daily observation, the observation is continued for 1 month, and the length of a new branch is investigated for the last time. Through experiments, the honeysuckle powder has obvious disease prevention and growth promotion effects, and the prevention and treatment effects and growth promotion effects on honeysuckle powdery mildew are shown in table 1.
Table 1 units of lesion change after bacterial liquid spraying: (cm)
The 50-time and 100-time dilution of the T79 bacterial liquid of the bacillus polymyxa has 100 percent of control effect on honeysuckle powdery mildew at 29 days, and the powdery mildew recrudescence starts at 23 days at 400 times, so that the disease course is not fast. The prevention and treatment effect is not ideal at 800 times, the disease spots start to grow slowly after the 8 th day, and the prevention and treatment effect is not ideal. The length of newly-grown branches of honeysuckle is measured on the 29 th day, and the growth degree of the branches of honeysuckle is found to have obvious dependence on the concentration of bacterial liquid, so that the bacillus polymyxa T79 bacterial liquid has obvious growth promoting effect. When this experiment was carried out, it was found that the test strain honeysuckle brown spot occurred very low, with a significant gap from the control, but no complete data was collected because it was not the main standard of the experiment. The strain sprayed with the bacillus polymyxa T79 bacterial liquid has bright leaf color and increased activity in a short time.
Carrying out rooting test on young branches of 50 times diluted water-cultured honeysuckle, glossy privet and buxus fortunei by using a bacillus polymyxa T79 bacterial solution, and carrying out comparison with clear water, wherein after 45 days, the activity of the honeysuckle branches is enhanced and root, the average length is 2.33cm, powdery mildew on leaves is caused, but the leaves are self-healing; only one of the clear water control groups had root, and the leaves were all on powdery mildew and dropped off. The glossy privet and the Chinese box leaves only grow callus, and are not different from the control. The bacillus polymyxa T79 is shown to have selectivity to honeysuckle.
At present, excessive use of traditional pesticides and fertilizers causes drug resistance of a plurality of plant diseases and insects, which causes vicious circle of ecological environment, and paenibacillus polymyxa can secrete a large amount of active substances, has inhibitory activity on a plurality of bacteria and fungi, can improve disease resistance of plants, promote plant growth and yield, and can generate polypeptide antibacterial substances with better antagonistic activity on plant pathogenic fungi. It is a better biocontrol bacterium. In addition, the bacillus polymyxa also has various biological activities such as enzyme activities, antioxidation, anti-tumor and the like, and when the culture of the bacillus polymyxa T79 is still relatively immature, the bacillus polymyxa T79 cannot be well cultured.
Disclosure of Invention
The invention aims at providing bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and a culture method thereof, aiming at the defects in the prior art.
In order to achieve the above purpose, the invention adopts the following technical scheme: the bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and a culture method thereof comprise the following specific steps: activating strains; culturing bacterial liquid; preparing a special culture medium: the special culture medium has nutrition supply capacity to the bacillus polymyxa T79 which exceeds that of an LB culture medium, and under the same culture condition, the content of the special culture medium is 6 times higher than that of the bacillus polymyxa T79; and (5) producing bacterial liquid or mother strain.
More specifically, the present invention is as follows: the specific operation of the strain activation is as follows: and (3) on a sterilized ultra-clean workbench, picking out the stored strains in the glycerol tube by using an inoculating needle, inoculating the stock seeds on a prepared LB plate medium by using a streaking method, and culturing in an incubator for a period of time, wherein the culturing time in the incubator is 48 hours.
More specifically, the present invention is as follows: the specific operation of the bacterial liquid culture is as follows: washing the bacterial colony on the flat-plate culture medium with sterile physiological saline into a triangular flask containing the sterilized LB liquid culture medium, packaging with a sealing film, and culturing for 48h at 200 rpm on a constant-temperature shaking table at 37 ℃ with the mass fraction of inoculation amount of 1/50.
More specifically, the present invention is as follows: the special culture medium preparation comprises a liquid culture medium formula and a solid culture medium formula, wherein the liquid culture medium formula specifically comprises the following components: 25g of soybean powder, 15.5g of corn powder, 17.5g of starch, 10g of calcium chloride and 1000ml of tap water, and sterilizing the mixture in an autoclave at 121 ℃ for 20 minutes, wherein the formula of the solid culture medium is specifically as follows; 25g of soybean powder, 15.5g of corn powder, 17.5g of starch, 1.6g of calcium chloride, 55ml of tap water and sterilizing the mixture in an autoclave at 121 ℃ for 20 minutes.
More specifically, the present invention is as follows: the bacterial liquid or mother strain production comprises the following specific steps: after the special culture medium is prepared, inoculating the bacillus polymyxa T79 strain activated by the LB culture medium on an ultra-clean workbench, wherein the inoculation mass ratio is 1:50, uniformly mixing the strain and the culture medium, wherein the liquid culture medium is cultured for 48 hours at a constant temperature of 37 ℃ in a shaking table at 200 rpm, and the solid culture medium is cultured for 3-5 days in a biochemical incubator at 37 ℃ so that the strain amount reaches 24 hundred million/g.
More specifically, the present invention is as follows: after the bacterial liquid or the mother strain is produced, bacterial powder extraction or metabolite extraction can be performed, and the bacterial liquid or the mother strain can also be used as bacterial fertilizer for open fermentation or industrial production mother strain, and the inoculation mass ratio is 1: 50-100.
After the technical scheme is adopted, the invention has the beneficial effects that: by adopting the culture method to culture the bacillus polymyxa T79, the bacillus polymyxa T79 can be cultured more stably, the culture efficiency is more efficient, and the bacillus polymyxa T79 can be better applied to different fields.
Detailed Description
The technical scheme adopted by the specific embodiment is as follows: the method comprises the following specific steps: activating strains; culturing bacterial liquid; preparing a special culture medium: the special culture medium has nutrition supply capacity to the bacillus polymyxa T79 which exceeds that of an LB culture medium, and under the same culture condition, the content of the special culture medium is 6 times higher than that of the bacillus polymyxa T79; and (5) producing bacterial liquid or mother strain.
The specific operation of the strain activation is as follows: on a sterilized ultra-clean workbench, picking out stored strains in a glycerol pipe by an inoculating needle, inoculating stock seeds on a manufactured LB plate culture medium by a streaking method, and culturing in an incubator for a period of time, wherein the culturing time in the incubator is 48 hours, and the specific operation of bacterial liquid culture is as follows: flushing bacterial colonies on a flat plate culture medium into a triangular flask containing a sterilized LB liquid culture medium by using sterile normal saline, packaging by using a sealing film, culturing for 48 hours at 200 rpm on a constant temperature shaking table at 37 ℃ with the mass fraction of inoculation amount of 1/50, and preparing a special culture medium comprising a liquid culture medium formula and a solid culture medium formula, wherein the liquid culture medium formula specifically comprises: 25g of soybean powder, 15.5g of corn powder, 17.5g of starch, 10g of calcium chloride and 1000ml of tap water, and sterilizing the mixture in an autoclave at 121 ℃ for 20 minutes, wherein the formula of the solid culture medium is specifically as follows; 25g of soybean powder, 15.5g of corn powder, 17.5g of starch, 1.6g of calcium chloride, 55ml of tap water and sterilizing for 20 minutes at 121 ℃ in an autoclave, wherein the specific steps of bacterial liquid or mother seed production are as follows: after the special culture medium is prepared, inoculating the bacillus polymyxa T79 strain activated by the LB culture medium on an ultra-clean workbench, wherein the inoculation mass ratio is 1:50, uniformly mixing the strain and the culture material, wherein a liquid culture medium is cultured for 48 hours at a constant temperature of 37 ℃ on a shaking table at 200 rpm, a solid culture medium is cultured for 3-5 days in a biochemical incubator at 37 ℃, the bacterial load reaches 24 hundred million/gram, bacterial powder extraction or metabolite extraction can be carried out after bacterial liquid or mother strain is produced, and the bacterial liquid or the mother strain can also be used as an open fermentation bacterial fertilizer or an industrial production mother strain, and the inoculation mass ratio is 1: 50-100.
After the technical scheme is adopted, the invention has the beneficial effects that: by adopting the culture method to culture the bacillus polymyxa T79, the bacillus polymyxa T79 can be cultured more stably, the culture efficiency is more efficient, and the bacillus polymyxa T79 can be better applied to different fields.
Note that: the 16s gene sequencing of B.polymyxa T79 was as follows:
the foregoing is merely illustrative of the present invention and not restrictive, and other modifications and equivalents thereof may occur to those skilled in the art without departing from the spirit and scope of the present invention.
Claims (10)
1. A bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and a culture method thereof are characterized in that: the method comprises the following specific steps:
1) Activating strains;
2) Culturing bacterial liquid;
3) Preparing a special culture medium: the special culture medium has nutrition supply capacity to the bacillus polymyxa T79 which exceeds that of an LB culture medium, and under the same culture condition, the content of the special culture medium is 6 times higher than that of the bacillus polymyxa T79;
4) And (5) producing bacterial liquid or mother strain.
2. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 1, wherein the bacillus polymyxa T79 is characterized in that: the specific operation of activating the strain in the step 1 is as follows: on a sterilized ultra-clean workbench, picking out the stored strains in the glycerol tube by using an inoculating needle, inoculating the stock strains on a prepared LB plate culture medium by using a streaking method, and culturing for a period of time in an incubator.
3. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 2, wherein the bacillus polymyxa T79 is characterized in that: the incubation time in the incubator was 48 hours.
4. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 1, wherein the bacillus polymyxa T79 is characterized in that: the specific operation of the bacterial liquid culture in the step 2 is as follows: and (3) flushing the bacterial colony on the flat-plate culture medium into a triangular flask containing the sterilized LB liquid culture medium by using sterile normal saline, and packaging by using a sealing film, wherein the mass fraction of the inoculation amount is 1/50.
5. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 4, wherein the method is characterized in that: after the packaging, the culture is carried out on a constant temperature shaking table at 37 ℃ for 48 hours at 200 rpm.
6. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 1, wherein the bacillus polymyxa T79 is characterized in that: the special culture medium preparation in the step 3 comprises a liquid culture medium formula and a solid culture medium formula.
7. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 6, wherein the method is characterized in that: the formula of the liquid culture medium specifically comprises the following components: 25g of soybean powder, 15.5g of corn powder, 17.5g of starch, 10g of calcium chloride and 1000ml of tap water, and sterilizing the mixture in an autoclave at 121 ℃ for 20 minutes.
8. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 6, wherein the method is characterized in that: the formula of the solid culture medium is specifically as follows; 25g of soybean powder, 15.5g of corn powder, 17.5g of starch, 1.6g of calcium chloride, 55ml of tap water and sterilizing the mixture in an autoclave at 121 ℃ for 20 minutes.
9. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 1, wherein the bacillus polymyxa T79 is characterized in that: the specific steps of the bacterial liquid or mother strain production in the step 4 are as follows: after the special culture medium is prepared, inoculating the bacillus polymyxa T79 strain activated by the LB culture medium on an ultra-clean workbench, wherein the inoculation mass ratio is 1:50, uniformly mixing the strain and the culture medium, wherein the liquid culture medium is cultured for 48 hours at a constant temperature of 37 ℃ in a shaking table at 200 rpm, and the solid culture medium is cultured for 3-5 days in a biochemical incubator at 37 ℃ so that the strain amount reaches 24 hundred million/g.
10. The bacillus polymyxa T79 with disease prevention and growth promotion effects on honeysuckle and the culture method thereof according to claim 1, wherein the bacillus polymyxa T79 is characterized in that: after the bacterial liquid or the mother strain is produced, bacterial powder extraction or metabolite extraction can be performed, and the bacterial liquid or the mother strain can also be used as bacterial fertilizer for open fermentation or industrial production mother strain, and the inoculation mass ratio is 1: 50-100.
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