CN110628685B - Bacillus subtilis strain and application thereof in agricultural production - Google Patents
Bacillus subtilis strain and application thereof in agricultural production Download PDFInfo
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Abstract
The invention relates to the technical field of functional microorganism screening and application, and particularly provides a novel bacillus subtilis VB7-2(Bacillus subtilis)Bacillus subtilis VB7-2), accession number CCTCC NO: m2019442 and provides application thereof in agricultural production. The bacillus subtilis is screened from potato planting area scab-infected land blocks in Keshan county of Heilongjiang province, has obvious inhibition effect on potato scab streptomyces, can effectively prevent and treat common crop diseases, improves crop yield and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of functional microorganism screening, in particular to a bacillus subtilis strain and application thereof in agricultural production.
Background
The microorganisms have a close relationship with agriculture, and in soil which is called 'microbial capital nutrition', the microorganisms play a main role in the qualitative circulation and have irreplaceable functions. They decompose the animal and plant residual wastes to convert them into humus, promoting the formation of a good structure of the soil. Many soil microorganisms can fix nitrogen in the air and convert various organic matters, and continuously provide various nutrient elements such as carbon, nitrogen, phosphorus, potassium and the like which can be effectively utilized for plants. Antagonistic microorganisms of pathogenic microorganisms of insects and plant germs are also widely present in nature and can be used for the control of plant diseases and insect pests to partially replace chemical pesticides. In addition, various products such as organic acid, amino acid, growth hormone, antibiotics, various enzyme preparations and the like can be produced through microbial propagation and fermentation, can be respectively used as feed additives, food additives, pesticides and the like, and are increasingly widely applied.
1. Microbial fertilizer
Microbial fertilizers, also known as bacterial fertilizers or biofertilizers, are a class of microbial preparations produced by artificially culturing microorganisms beneficial to plants. The microbial fertilizer has comprehensive effect, does not directly provide nutrient elements for crops generally, has indirect nutrition effect, and can improve soil fertility, improve soil structure, stimulate crop growth, improve crop quality, and enhance disease resistance (pest) and stress resistance of plants. After the microbial fertilizer is used, the application amount of the fertilizer can be effectively reduced, and the utilization rate of the fertilizer is improved.
There are five major groups according to the microbial species: bacterial fertilizers (such as rhizobium fertilizer, azotobacter fertilizer, phosphate solubilizing bacterial fertilizer, potassium solubilizing bacterial fertilizer and photosynthetic bacterial fertilizer); ② actinomycete fertilizer (such as antibiotic fertilizer); thirdly, fungus fertilizer (mycorrhizal fungus fertilizer, including ectomycorrhizal fungus agent and endophytic fungus agent); algae fertilizer (such as nitrogen-fixing blue algae bacterial fertilizer); compound microbial fertilizer, i.e. the fertilizer is formed by combining more than two microbes according to a certain proportion.
2. Microbial pesticide
The microbial pesticide is a non-chemically synthesized microbial preparation with the functions of killing insects and preventing diseases, such as a microbial insecticide, a bactericide, an agricultural antibiotic and the like, and the microorganisms comprise bacteria, viruses and fungi for killing insects and preventing diseases.
The most studied and used amount of the insecticidal microorganisms is bacillus thuringiensis, and one or more protein crystals called endotoxin with strong bactericidal action can be formed in the bacillus during spore generation, so that the bacillus thuringiensis can be widely used for moisture prevention of grains, economic crops, vegetables, forestry and certain sanitary pests. The bacillus thuringiensis has the other outstanding advantage of strong selectivity, is very safe to people, livestock, natural enemies and plants, and can be called as 'pollution-free pesticide'. However, the bacterium also has some disadvantages and shortcomings, such as the toxin protein crystal is easy to be decomposed by the effect of environmental factors; the insecticidal action is not lasting, and the field control effect can only be maintained for 3 to 4 days; the insecticidal spectrum is narrow, the insecticidal composition is only effective to partial lepidoptera pests, and the pests may generate drug resistance after being used for a long time.
In addition, many microorganisms in nature have inhibitory and antagonistic effects on plant pathogens. For example, many bacteria, actinomycetes and fungi prevent the occurrence and spread of diseases by producing antibiotics or competing for nutrition and living spaces, but the effect of controlling diseases is also affected by complicated environmental conditions and cannot be stabilized or sustained.
3. Microbial preparation for feed
The research of novel feed microbial preparation becomes a main means for feed industry technical innovation and product updating, and plays an important role in eliminating anti-nutritional factors, improving resource utilization rate, opening up new feed sources and solving the environmental pollution of animal husbandry.
4. Environmental decontamination
Microorganisms can play a unique role in the decomposition of various agricultural environmental pollutants, and microbial strains capable of effectively decomposing various pollutants such as plant straws, livestock and poultry manure, residual pesticides, industrial wastewater, household garbage and the like are continuously developed and applied. The biodegradation and biological recovery technology has the obvious advantages of high efficiency, high speed, low cost, mild reaction condition, no secondary pollution and the like, and becomes an important development direction for treating agricultural environmental pollution internationally. Due to the complexity of pollutant components and environmental pollution factors, microorganisms used for environmental pollution purification should have not only strong degradability and environmental tolerance, but also diversity of degradability.
The wide application of the microorganism in agricultural production is beneficial to improving the agricultural ecological environment, increasing the grain yield, reducing the environmental pollution, realizing the sustainable development of agriculture, and has obvious practical significance and profound historical influence on promoting the revolution of agricultural production. Digging, screening and expanding new functional microbial strains, breeding microbial products with excellent crop affinity, regionality, pertinence, adaptability and productivity, and improving the stability of application effect, and is the research focus in the field at present.
Disclosure of Invention
The invention aims to provide a novel Bacillus subtilis strain and application thereof in agricultural production. The bacillus subtilis is screened from potato planting area scab-infected land blocks in Keshan county of Heilongjiang province, has obvious inhibition effect on potato scab streptomyces, can effectively prevent and treat common crop diseases, improves crop yield and has wide application prospect.
The invention provides a Bacillus subtilis named as Bacillus subtilis VB7-2(Bacillus subtilis VB7-2), which is preserved in China center for type culture Collection of Wuhan university in Wuhan, China in 6 months and 6 days in 2019, wherein the preservation number is CCTCC NO: and M2019442.
On the one hand, the invention provides the application of the bacillus subtilis in plant disease control.
The plant diseases comprise any one of anthracnose, banded sclerotial blight, curvularia, root rot, southern blight, powdery mildew, scab, rotten fruit disease, gray mold, fusarium wilt, brown spot and fruit rot.
The invention provides an application of the bacillus subtilis in a biological fertilizer.
The invention also provides a microbial preparation which comprises the bacillus subtilis VB 7-2.
The microbial preparation also comprises any one or the combination of two or more of bacillus, pseudomonas, agrobacterium, azotobacter, rhizobium, penicillium, aspergillus, rhizopus and streptomyces.
The viable bacteria content of the bacillus subtilis VB7-2 in the microbial preparation is at least 108CFU/g。
The invention also provides application of the microbial preparation in a biological fertilizer.
The invention also provides application of the microbial preparation in plant disease control.
The screened bacillus subtilis VB7-2 has a strong inhibiting effect on the potato scab streptomyces, and the width of a bacteriostatic zone exceeds 23 mm. The bacillus subtilis can be widely applied to prevention and control of common plant diseases, wherein the prevention and control efficiency of potato scab and peanut rotten fruit diseases is respectively up to 76.5% and 87.1%, and unexpected technical effects are achieved.
The bacillus subtilis VB7-2 can obviously improve the soil nutrient structure and improve the soil fertility, and the contents of alkaline hydrolysis nitrogen, available phosphorus and quick-acting potassium in the soil of the treatment group applying the bacillus subtilis VB7-2 are respectively improved by 123.8 percent, 443.5 percent and 279.7 percent compared with the control group. The strain can also obviously promote the growth of the corn, improve the production performance of the corn, generally improve the corn yield by 14.2-23.19 percent and have obvious yield-increasing effect. The bacillus subtilis VB7-2 can also obviously improve the yield of the melons and improve the quality of the melons. Compared with a control group, the yield of the melons in the treatment group is generally increased by 11.0-20.3%, and the yield increase effect is obvious; the content of soluble solids in the processed muskmelon is improved by 11.0-23.2 percent, the content of vitamin C is improved by 12.7-35.2 percent, and unexpected technical effects are achieved.
In conclusion, the bacillus subtilis VB7-2 provided by the invention can be used as a biological control agent, a biological fertilizer and the like, is widely applied to the field of agricultural production, and has obvious effect and wide application prospect.
Detailed Description
The equipment and reagents used in the examples of the present invention may be selected from any commercially available ones. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.
The culture medium formulation involved for the present invention is as follows:
YME solid plate: 4g of yeast powder, 10g of maltose extract, 4g of glucose dextrose, 15g of agar and distilled water, wherein the volume is constant to 1L, the pH value is 7.2, moist heat sterilization is carried out at 121 ℃ for 15min, and the mixture is cooled and poured into a culture dish.
LB solid plate: 10g of peptone, 5g of yeast powder, 10g of NaCl, 15g of agar and distilled water, wherein the volume is constant to 1L, the pH value is 7.0, moist heat sterilization is carried out at 121 ℃ for 15min, and the mixture is cooled and poured into a culture dish.
The invention is further illustrated by the following specific examples.
Example 1 screening of microorganisms in soil
1. Separating and purifying soil microorganisms:
(1) soil sample: collected from potato planting areas of Keshan county of Heilongjiang province, wherein the potato planting areas are scab-affected plots.
(2) 0.5g of a soil sample was weighed and dissolved in 4.5ml of sterile water to make 1: 10, then sucking 0.5ml of the soil solution from the soil solution and putting the soil solution into 4.5ml of sterile water to prepare a mixture of 1: 100, by analogy with this method, 1: 106-107The soil dilution solution of (1).
Taking 0.1ml of 3-4 diluents with proper concentration and uniformly coating the diluents on an LB solid plate; culturing at 37 deg.C for 2d, taking out, selecting single colony, and continuously separating and purifying until the colony grown on each plate has consistent shape and color, to obtain single strain.
By the above method, the applicant selected 10 strains of bacteria, which were designated as MC1, MC2, MC3, … … and MC 10.
2. Biocontrol bacteria primary screen
Respectively inoculating the 10 separated and purified strains into nutrient broth culture media, and culturing at 37 ℃ and 220r/min for 14h to prepare test bacterial liquid for later use.
Pathogenic bacteria of potato streptomyces scabicus (purchased from China general microbiological culture collection center) is prepared into bacterial suspension, and 100 mu L of bacterial suspension is coated on a YME solid plate; respectively drawing the bacterial liquid of the 10 strains on YME solid plates coated with streptomyces scabiosus; and (3) carrying out inverted static culture at 28 ℃ for 2-3 days, and judging the bacteriostatic effect according to the size of the generated bacteriostatic zone. Specific results are shown in table 1.
TABLE 1 inhibitory Effect of different strains on Streptomyces solani
As can be seen from the data in Table 1, the strains selected from the soil according to the present invention have the strongest inhibitory effects on Streptomyces solani of MC3, MC7, MC9 and MC 10. Wherein, the MC7 strain has the best bacteriostatic effect, and the width of the bacteriostatic zone exceeds 23 mm.
Example 2 prevention and treatment of common scab in Potato
1. Preparation of fungal powder
Respectively carrying out liquid fermentation on MC3, MC7, MC9 and MC10 in a 3-ton fermentation tank, and stopping fermentation when the microscopic spore rate reaches more than 90%; centrifuging at 5000rpm for 10min, removing supernatant, and spray drying to obtain powder with viable bacteria amount of 10 hundred million/g.
2. Prevention and treatment experiment for potato scab
The experimental site: in the wooden towns in Tengzhou city, the soil type is brown soil.
The experiment is provided with 30 experiment areas, and each experiment area is 110m in area2And 5 meters of separation was maintained between each experimental zone. 3 experimental zones were randomly selected for each treatment group.
(1) Blank control group: no bacterial powder was applied;
(2) fungus powder treatment group: two fertilization treatment modes of seed dressing and basal application are respectively adopted, and the using amount of the fungus powder (the living fungus amount is 10 hundred million/g)) is 3 kg/mu.
Treatment group 1: cutting potato seeds into blocks, adding MC3 strain powder, and sowing;
treatment group 2: cutting potato seeds into blocks, adding MC7 strain powder, and sowing;
treatment group 3: cutting potato seeds into blocks, adding MC9 strain powder, and sowing;
treatment group 4: cutting potato seeds into blocks, adding MC10 strain powder, and sowing;
treatment group 5: basal application of MC3 strain powder;
treatment group 6: basal application of MC7 strain powder;
treatment group 7: basal application of MC9 strain powder;
treatment group 8: applying MC10 bacterial powder.
In the potato harvesting period, counting the incidence of potato scab of each group, and calculating disease index and prevention and treatment efficiency; and simultaneously, the physical and chemical property indexes of each group of soil are comprehensively evaluated, and specific results are shown in tables 2 and 3.
The disease index is [ Σ (disease level value × number of diseased leaves at this level)/(total number of investigated leaves × highest disease level value) ] × 100.
The calculation formula of the prevention and treatment efficiency is [1- (disease index of treatment group/disease index of control group) ] × 100%.
TABLE 2 influence of different bacterial powders and application modes on control effect of potato scab
As can be seen from the data in Table 2, the four strains of MC3, MC7, MC9 and MC10 screened by the invention have obvious control effect on potato scab, and the effect of the basal dressing bacterial powder is generally superior to that of seed dressing. The MC7 strain has the best prevention and treatment effect on potato scab, the prevention and treatment efficiency can reach 76.5% when basal application is carried out, and unexpected technical effect is achieved.
TABLE 3 influence of different bacterial powders and application modes on soil physicochemical index and potato yield
As can be seen from the data in Table 3, the contents of alkaline-hydrolyzable nitrogen, available phosphorus and available potassium in the soil of each treatment group are significantly improved by applying the powder of four strains of MC3, MC7, MC9 and MC10 screened by the present invention compared with the control group. The content of nitrogen, phosphorus and potassium in the soil of the treatment group 6 which is subjected to basal application of the MC7 microbial inoculum is the highest, and is respectively improved by 123.8%, 443.5% and 279.7% compared with that of the control group. Therefore, the MC7 bacterial strain can effectively improve the nitrogen, phosphorus and potassium content in the soil, improve the soil nutrition structure, obviously improve the soil fertility and obtain unexpected technical effects.
Example 3 identification of MC7 Strain
The MC7 strain is subjected to molecular biological identification, the 16S rDNA sequence of the strain is measured, and Blast comparison is carried out in a GenBank nucleic acid database. Combining the biological characteristics of the MC7 strain and the 16S rDNA comparison result, the applicant confirms that the MC7 strain is Bacillus subtilis and is named as Bacillus subtilis VB7-2(Bacillus subtilis VB 7-2).
The applicant has already preserved the Bacillus subtilis VB7-2(Bacillus subtilis VB7-2) in the China center for type culture collection of the university of Wuhan, China at 6.6.2019 with the preservation number of CCTCC M2019442.
Example 4 application of Bacillus subtilis VB7-2 in prevention and treatment of peanut rot disease
1. The experimental site:
the Qingdao city is a large-field planting area of the peanuts in the seven-grade Zhenxiao town, and the land condition in the planting area is uniform.
2. Experiment for preventing and treating rotten fruit disease of peanut:
a square area of 10m multiplied by 10m is selected as an experimental area, 12 experimental areas are arranged in total, and an interval of 5 meters is kept between each two experimental areas. 3 experimental zones were randomly selected for each treatment group.
(1) Blank control group: no bacterial powder is applied, only organic fertilizer is applied in a scattering way, the using amount is 50 kg/mu, and then the soil is turned over and peanuts are sowed;
(2) VB7-2 bacterial powder treatment group: the bacillus subtilis VB7-2 powder (the viable count is 10 hundred million/g)) and an organic fertilizer are scattered on the ground, the dosage of the organic fertilizer is 50 kg/mu, and then the peanut is sowed after the soil is turned over. Wherein:
treatment group 1: the consumption of VB7-2 bacterial powder is 1 kg/mu;
treatment group 2: the consumption of VB7-2 bacterial powder is 3 kg/mu;
treatment group 3: the consumption of VB7-2 bacterial powder is 5 kg/mu.
In the peanut harvesting period, the rotten fruit number of each group of peanuts is respectively counted, the disease index and the control efficiency of the bacillus subtilis VB7-2 on the rotten fruit diseases of the peanuts are calculated, and specific results are shown in table 4.
The disease index is [ Σ (disease level value × number of diseased leaves at this level)/(total number of investigated leaves × highest disease level value) ] × 100.
The calculation formula of the prevention and treatment efficiency is [1- (disease index of treatment group/disease index of control group) ] × 100%.
TABLE 4 prevention and treatment effects of Bacillus subtilis VB7-2 on peanut rot fruit diseases
As can be seen from the data in Table 4, the indexes of the rot disease of peanuts in each treatment group which is applied with the powder of the Bacillus subtilis VB7-2 are only 9.3-23.5, which is far lower than that of the control group. Therefore, the bacillus subtilis VB7-2 provided by the invention has an obvious prevention and treatment effect on the peanut rot fruit diseases, the prevention and treatment efficiency is up to 87.1 percent at most, and an unexpected technical effect is achieved.
Example 5 application of Bacillus subtilis VB7-2 in corn planting
1. The experimental site:
the Qingdao city is a maize field planting area of the seven-grade Zhenjin, and the land condition in the planting area is uniform.
2. Corn planting experiment:
a10 m20 m area is selected as an experimental area, 20 experimental areas are arranged in total, and the interval of 1 meter is kept between every two experimental areas. Each treatment group randomly selected 5 experimental zones for the experiment.
(1) Blank control group: no bacterial powder is applied, only organic fertilizer is applied in a scattering way, the using amount is 50 kg/mu, and then the soil is turned over and corn is sown;
(2) VB7-2 bacterial powder treatment group: the method comprises the steps of scattering bacillus subtilis VB7-2 powder (the viable count is 10 hundred million/g) and an organic fertilizer on the ground, wherein the using amount of the organic fertilizer is 50 kg/mu, then turning the ground and sowing corn. Wherein:
treatment group 1: the consumption of VB7-2 bacterial powder is 1 kg/mu;
treatment group 2: the consumption of VB7-2 bacterial powder is 3 kg/mu;
treatment group 3: the consumption of VB7-2 bacterial powder is 5 kg/mu.
In the mature period of the corns, the corns are harvested, threshed and aired in a unified mode, the yield of the corns in each experimental area is counted, the average yield and the yield increasing rate of the corns are calculated, and specific results are shown in a table 5.
Yield increase is (corn yield of treatment group-corn yield of blank control group)/corn yield of blank control group x 100%.
TABLE 5 influence of Bacillus subtilis VB7-2 on maize yield
| Experiment grouping | Average yield kg/mu | Increase of yield |
| Control group | 601.00 | - |
| VB7-2 treatment group 1 | 686.34 | 14.20％ |
| VB7-2 treatment group 2 | 724.51 | 20.55％ |
| VB7-2 treatment group 3 | 740.37 | 23.19％ |
The results in Table 5 show that the yield of the treated corn can be generally improved by 14.2-23.19% by applying the powder of the Bacillus subtilis VB7-2 in the seedling stage of the wheat, and the yield increasing effect is obvious. Therefore, the bacillus subtilis VB7-2 provided by the invention can obviously promote the growth of corn, improve the production performance of the corn, further increase the yield of the corn and obtain obvious effects.
Example 6 application of Bacillus subtilis VB7-2 in melon planting
1. The experimental site:
big-arch shelter is planted to chinese countryside melon in lai xi city of Qingdao.
2. Experiment design:
a10 m multiplied by 8m area is selected as an experimental area, and 10 ridges of melons are arranged in each experimental area, and about 400 +/-10 plants are obtained. The total number of the experimental areas is 12, and a protection row is arranged between each experimental area. 3 experimental zones were randomly selected for each treatment group.
(1) Blank control group: watering the roots of the sweet melon seedlings with clear water;
(2) VB7-2 bacterial powder treatment group: VB7-2 strain powder (the viable strain amount is 10 hundred million/g) is applied with water after the melon seedlings are transplanted according to the dosage of 1-5 kg/mu, and the application is carried out once every 7 days and is continuously carried out for three times. Wherein:
treatment group 1: the consumption of VB7-2 bacterial powder is 1 kg/mu;
treatment group 2: the consumption of VB7-2 bacterial powder is 3 kg/mu;
treatment group 3: the consumption of VB7-2 bacterial powder is 5 kg/mu.
After the muskmelon is ripe, centrally picking the muskmelons with the maturity of more than 8 days at 6:00-8:00 a day, weighing, and recording the daily output of the muskmelons in each experimental area; 20 melons were randomly picked from the daily melons picked in each experimental area, and the soluble solid content (mass%) and the vitamin C content (mg/100g) were measured.
After all melons are harvested, the total yield of the melons in each experimental area is counted, the average total yield and the yield increase rate of the melons in the control group and the melons in the treatment group are calculated respectively, and specific results are shown in table 6; the average content of soluble solids and vitamin C in the melons of the control group and the treated group were calculated, and the specific results are shown in table 7.
Yield increase is (melon yield in treatment group-melon yield in blank control group)/melon yield in blank control group x 100%.
TABLE 6 influence of Bacillus subtilis VB7-2 on melon yield
| Experiment grouping | Average yield kg/mu | Increased yield% |
| Blank control group | 2315 | - |
| VB7-2 treatment group 1 | 2568.7 | 11.0％ |
| VB7-2 treatment group 2 | 2654.6 | 14.7％ |
| VB7-2 treatment group 3 | 2783.8 | 20.3％ |
TABLE 7 influence of Bacillus subtilis VB7-2 on melon quality
As can be seen from the data in tables 6 and 7, the yield of the melon can be remarkably improved and the quality of the melon can be improved by applying the powder of the Bacillus subtilis VB7-2 in the seedling stage of the melon. Compared with a control group, the yield of the melons in the treatment group is generally increased by 11.0-20.3%, and the yield increase effect is obvious; the content of soluble solids in the processed muskmelon is improved by 11.0-23.2 percent, the content of vitamin C is improved by 12.7-35.2 percent, and unexpected technical effects are achieved.
In conclusion, the bacillus subtilis VB7-2 provided by the invention can be independently used as a biocontrol microbial inoculum, a biological fertilizer and the like for controlling plant diseases such as potato scab, peanut rotten fruit disease and the like, is widely applied to the field of agricultural production, can be combined with any one or more of other bacillus, pseudomonas, agrobacterium tumefaciens, azotobacter, rhizobium, penicillium, aspergillus, rhizopus and streptomyces, is used for controlling other common plant diseases, has the control efficiency of more than 66 percent, has the growth-promoting and yield-increasing effects of more than 18 percent, has obvious effect and wide application prospect.
Claims (7)
1. Bacillus subtilis (B.subtilis)Bacillus subtilis) VB7-2, wherein the preservation number of the bacillus subtilis is CCTCC NO: and M2019442.
2. The use of the bacillus subtilis of claim 1 for controlling a plant disease, wherein the plant disease is potato scab or peanut rotten fruit disease.
3. Use of the bacillus subtilis of claim 1 for the preparation of a biological fertilizer.
4. A microbial preparation comprising the bacillus subtilis of claim 1.
5. The microbial preparation of claim 4, wherein the microbial preparation has a viable count of Bacillus subtilis of at least 108CFU/g。
6. Use of the microbial preparation according to claim 4 or 5 for the control of plant diseases, wherein the plant diseases are potato scab or peanut rotten fruit diseases.
7. Use of a microbial preparation according to claim 4 or 5 in the preparation of a biological fertilizer.
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Citations (5)
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| KR101740545B1 (en) * | 2015-06-09 | 2017-05-29 | 대한민국 | Bacillus subtilis strain YD-6 and uses thereof |
| KR20170130876A (en) * | 2016-05-19 | 2017-11-29 | 이광수 | Composition for Controlling Strawberry Pathogen Comprising Bacillus subtilis FNR-10 As an Active Ingredient |
| CN108865949A (en) * | 2018-07-20 | 2018-11-23 | 北京泰克美高新技术有限公司 | Bacillus subtilis TKM-1 and its application |
| CN109320363A (en) * | 2018-12-07 | 2019-02-12 | 广东植物龙生物技术股份有限公司 | A kind of bacillus liquid bluk recombination microbial-bacterial fertilizer and preparation method thereof |
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