CN104560769A - Microbial agent as well as preparation method and application thereof - Google Patents
Microbial agent as well as preparation method and application thereof Download PDFInfo
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- CN104560769A CN104560769A CN201310516573.5A CN201310516573A CN104560769A CN 104560769 A CN104560769 A CN 104560769A CN 201310516573 A CN201310516573 A CN 201310516573A CN 104560769 A CN104560769 A CN 104560769A
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Abstract
The invention provides a microbial agent. The microbial agent comprises a culture medium and thalli, wherein the thalli comprise Bacillus subtilis, Bacillus amyloliquefaciens and Bacillus safensis. The microbial agent provided by the invention can obtain a good plant seedling index and further improve yield and quality of vegetables, thereby having broad application prospects in the field of vegetable seedling raising. In addition, by using the microbial agent provided by the invention, a plant rhizosphere microbial environment can be improved, and beneficial bacteria are increased, thereby inhibiting the growth of harmful bacteria and further improving the disease resistance of plants.
Description
Technical field
The present invention relates to a kind of microbial bacterial agent and its preparation method and application; Or rather, relate to a kind of microbial bacterial agent and preparation method thereof, also relate to the application of described bacteria agent in grow seedlings of vegetable.
Background technology
Along with the development of world's agricultural economy, agricultural planting structure also experiencings corresponding adjustment, changed to agricultural modernization gradually by traditional farming, the cultivated area of vegetables also increases year by year, nursery is then a very important sport technique segment in vegetable growing, grow seedlings of vegetable not only can make vegetables listing ahead of time, can also improve yield of vegetables and improve quality.
Current grow seedlings of vegetable Problems existing is that grow seedlings of vegetable soil-borne disease is serious, and harmful bacteria amount reproduction, has a strong impact on grow seedlings of vegetable survival rate; Modern agriculture facility cultivation causes vegetable continuous cropping to be hindered more and more seriously simultaneously, and soil easily hardens, poor air permeability, and soil moisture content is poor, thus affects yield of vegetables, quality; Therefore, in the urgent need to developing the nursery correlation technique that can improve plant seedling vigorous index.
Summary of the invention
The object of the invention is to overcome the problem that the plant seedling vigorous index that exists in current grow seedlings of vegetable process is low, provide a kind of and can improve microbial bacterial agent of plant seedling vigorous index and its preparation method and application.
In order to realize first goal of the invention, the invention provides a kind of microbial bacterial agent, this microbial bacterial agent comprises medium and thalline, wherein, described thalline comprises bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and husky good fortune bacillus (Bacillus safensis).
In order to realize second goal of the invention, present invention also offers a kind of preparation method of microbial bacterial agent, wherein, the method comprises: the method comprises: bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and husky good fortune bacillus (Bacillus safensis) are inoculated in medium and are cultivated.
Present invention also offers described microbial bacterial agent in the application of described microbial bacterial agent in grow seedlings of vegetable.
Microbial bacterial agent provided by the invention can improve grow seedlings of vegetable survival rate, and such as, the result detected in embodiment 5-8 can be found out, although emergence rate respectively processed and do not have notable difference with contrasting seedling stage; But seedling vigorous index A3 is up to 0.325, contrasting the poorest is 0.077, each process with contrast between reach difference pole significance level, between respectively processing, A2, A3, A4 and A1 reach significance level, do not have difference between A2, A3, A4; Incidence of disease contrast is up to 5%, each process not morbidity; After watermelon seedlings field planting to land for growing field crops, flowering stage is A2 the earliest, and being August 1, is secondly A1, A2, and being contrast the latest, is August 10, illustrates that using microbial bacterial agent nursery to improve yields positive results; Gross yield A3 is up to 1441 kilograms, and minimum is contrast, 1092 kilograms, and each process reaches significance level with contrasting, and does not have difference between process; Quality of watermelon, pol is up to A3 and A4, and minimum is contrast; Watermelon late onset rate is up to contrast, reaches 13.33%, and minimum is A3 and A4, and morbidity Major Diseases is fusarium wilt; Comprehensively use microbial bacterial agent of the present invention can obtain good stand seedling vigorous index as can be seen from above, thus make the aspects such as final yield and quality all obviously be better than contrast, have broad application prospects in grow seedlings of vegetable field.In addition, owing to using microbial bacterial agent provided by the invention can improve plant rhizospheric microorganism environment, beneficial bacterium increases thus suppresses the growth of harmful bacteria, thus can also improve the disease resistance of plant.
Embodiment
The invention provides a kind of microbial bacterial agent, this microbial bacterial agent comprises medium and thalline, wherein, described thalline comprises bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and husky good fortune bacillus (Bacillus safensis).
The amount of the thalline contained in described microbial bacterial agent can in very large range change, and under preferable case, the total viable count contained by every gram of described microbial bacterial agent can be 2-10 × 10
7individual.
Preferably, in described microbial bacterial agent, with the total viable count in described microbial bacterial agent for benchmark, the viable count of bacillus subtilis (Bacillus subtilis) can be the 10-50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) can be able to be the 10-50% of total viable count for the viable count of the 10-50% of total viable count, husky good fortune bacillus (Bacillus safensis).
More preferably, with the total viable count in described microbial bacterial agent for benchmark, the viable count of bacillus subtilis (Bacillus subtilis) can be the 40-50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) can be able to be the 15-35% of total viable count for the viable count of the 15-35% of total viable count, husky good fortune bacillus (Bacillus safensis).
According to the present invention, the kind of described medium can in very large range change, the medium cultivating bacillus subtilis, bacillus licheniformis, bacillus amyloliquefaciens, husky good fortune bacillus can be can be used in for various, such as, can be the conventional medium such as beef-protein medium, broth bouillon, LB medium as seed culture medium, for the preservation of bacterial classification; And the medium of fermentation is generally used for production, the kind of these medium is also conventionally known to one of skill in the art.Above-mentioned medium can be commercially available or prepare according to the record of " microbiological culture media handbook " (Microbiology Culture Media Manual).Such as, beef-protein medium: beef extract 3g, peptone 10g, sodium chloride 5g, agar 15-20g, distilled water 1000ml, pH value 7.0-7.2,121 DEG C of sterilizing 20min; Fermentation medium: glucose 15g, starch 1g, beancake powder 25g, manganese sulphate 1g, potassium dihydrogen phosphate 1.5g, magnesium sulfate 0.5g, yeast extract 0.2g, iron chloride 0.1g, calcium carbonate 0.1g, pH value 7.0-7.2,121 DEG C of sterilizing 20min.
Present invention also offers the preparation method of described microbial bacterial agent, wherein, the method comprises: bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and husky good fortune bacillus (Bacillus safensis) are inoculated in medium and are cultivated.
According to the present invention, the method of described cultivation comprises: in respective independently cultivating system, cultivating bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and husky good fortune bacillus (Bacillus safensis) respectively, and proportionally mixing cultivating the microorganism obtained respectively.Under preferable case, by the cultivation in respective independently cultivating system and the mixing after cultivating, total viable count of the every gram of microbial bacterial agent obtained is made to be 2-10 × 10
7individual.
In a preferred embodiment, by in the cultivation separately independently in cultivating system and mixing proportionally after cultivating, the condition of described mixing has no particular limits, as long as the microbial bacterial agent obtained can be made to meet the following conditions: total to make viable count in described microbial bacterial agent for benchmark, the viable count of bacillus subtilis (Bacillus subtilis) is the 10-50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is the 10-50% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is the 10-50% of total viable count, preferably, with the total viable count in described microbial bacterial agent for benchmark, the viable count of bacillus subtilis (Bacillus subtilis) is the 40-50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is the 15-35% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is the 15-35% of total viable count.
According to the present invention, the bacterial classification of described bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), husky good fortune bacillus (Bacillus safensis) can be commercially available, such as, the husky good fortune bacillus (CCTCCAB205598) of the bacillus subtilis (ACCC01266) of Chinese agriculture Culture Collection preservation, bacillus amyloliquefaciens (ACCC1855), Beijing North Na Chuanlian Bioteknologisk Institut.
According to the present invention, the cultural method of described bacillus subtilis (Bacillus subtilis) has no particular limits as conventionally known to one of skill in the art, such as, can at the medium of 100 weight portions (glucose 5g, dregs of beans 30g, peptone 2g, distilled water 1000ml, PH7.0-7.2) bacterial strain of the bacillus subtilis (Bacillus subtilis) of inoculation 2-5 weight portion in, 37 DEG C of cultivations, until the viable count of bacillus subtilis (Bacillus subtilis) is 2-10 × 10
7individual/gram medium.
The cultural method of described bacillus amyloliquefaciens (Bacillus amyloliquefaciens) has no particular limits, such as, can at 100 weight portion medium (beancake powder 50g, sucrose 20g, ammonium sulfate 5g, trisodium citrate 2.5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate 0.5g, ferrous sulfate 0.05g, PH7.0-7.2) bacterial strain of the bacillus amyloliquefaciens (Bacillus amyloliquefaciens) of inoculation 2-5 weight portion in, 37 DEG C of cultivations, until the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is 2-10 × 10
7individual/gram medium.
The cultural method of described husky good fortune bacillus (Bacillus safensis) has no particular limits, such as, can at the medium of 100 weight portions (glucose 15g, starch 1g, beancake powder 25g, manganese sulphate 1g, potassium dihydrogen phosphate 1.5g, magnesium sulfate 0.5g, yeast extract 0.2g, iron chloride 0.1g, calcium carbonate 0.1g, PH7.0-7.2) bacterial strain of the husky good fortune bacillus (Bacillus safensis) of inoculation 2-5 weight portion in, 37 DEG C of cultivations, until the viable count of husky good fortune bacillus (Bacillus safensis) is 2-10 × 10
7individual/gram medium.
Present invention also offers the application of described microbial bacterial agent in grow seedlings of vegetable.
Described vegetables can be watermelon, muskmelon, cucumber, tomato, capsicum, eggplant etc., are preferably on the vegetables economic crops such as watermelon, muskmelon, cucumber.
Below by specific embodiment, further description is carried out to the present invention.
Embodiment 1
(1) at medium (the glucose 5g of 100 weight portions, dregs of beans 30g, peptone 2g, distilled water 1000ml, PH7.0-7.2) bacillus subtilis (ACCC01266) of inoculation 5 weight portions in, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of bacillus subtilis is 2 × 10
7individual/gram medium.
(2) at medium (the beancake powder 50g of 100 weight portions, sucrose 20g, ammonium sulfate 5g, trisodium citrate 2.5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate 0.5g, ferrous sulfate 0.05g, PH7.0-7.2) inoculate the bacillus amyloliquefaciens (ACCC1855) of 4 weight portions, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of bacillus amyloliquefaciens is 2 × 10
7individual/gram medium.
(3) at medium (glucose 15g, starch 1g, the beancake powder 25g of 100 weight portions, manganese sulphate 1g, potassium dihydrogen phosphate 1.5g, magnesium sulfate 0.5g, yeast extract 0.2g, iron chloride 0.1g, calcium carbonate 0.1g, PH7.0-7.2) the middle husky good fortune bacillus (CCTCCAB205598) inoculating 5 weight portions, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of husky good fortune bacillus is 2 × 10
7individual/gram medium.
(4) bacillus subtilis (Bacillus subtilis) will obtained respectively in step (1) to (3), , bacillus amyloliquefaciens (Bacillus amyloliquefaciens), husky good fortune bacillus (Bacillus safensis) proportionally mixes, make in the microbial bacterial agent obtained, the viable count of bacillus subtilis (Bacillus subtilis) is 20% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is 40% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is 40% of total viable count, obtain microbial bacterial agent A1.
Embodiment 2
(1) at medium (the glucose 5g of 100 weight portions, dregs of beans 30g, peptone 2g, distilled water 1000ml, PH7.0-7.2) bacillus subtilis (ACCC01266) of inoculation 4 weight portions in, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of bacillus subtilis is 8 × 10
7individual/gram medium.
(2) at medium (the beancake powder 50g of 100 weight portions, sucrose 20g, ammonium sulfate 5g, trisodium citrate 2.5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate 0.5g, ferrous sulfate 0.05g, PH7.0-7.2) the middle bacillus amyloliquefaciens (ACCC1855) inoculating 3 weight portions, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of bacillus amyloliquefaciens is 8 × 10
7individual/gram medium.
(3) at medium (glucose 15g, starch 1g, the beancake powder 25g of 100 weight portions, manganese sulphate 1g, potassium dihydrogen phosphate 1.5g, magnesium sulfate 0.5g, yeast extract 0.2g, iron chloride 0.1g, calcium carbonate 0.1g, PH7.0-7.2) the middle husky good fortune bacillus (CCTCCAB205598) inoculating 4 weight portions, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of husky good fortune bacillus is 8 × 10
7individual/gram medium.
(4) bacillus subtilis (Bacillus subtilis) will obtained respectively in step (1) to (3), , bacillus amyloliquefaciens (Bacillus amyloliquefaciens), husky good fortune bacillus (Bacillus safensis) proportionally mixes, make in the microbial bacterial agent obtained, the viable count of bacillus subtilis (Bacillus subtilis) is 35% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is 35% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is 30% of total viable count, obtain microbial bacterial agent A2.
Embodiment 3
(1) at medium (the glucose 5g of 100 weight portions, dregs of beans 30g, peptone 2g, distilled water 1000ml, PH7.0-7.2) bacillus subtilis (ACCC01266) of inoculation 3 weight portions in, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of bacillus subtilis is 5 × 10
7individual/gram medium.
(2) at medium (the beancake powder 50g of 100 weight portions, sucrose 20g, ammonium sulfate 5g, trisodium citrate 2.5g, potassium dihydrogen phosphate 0.3g, magnesium sulfate 0.5g, ferrous sulfate 0.05g, PH7.0-7.2) the middle bacillus amyloliquefaciens (ACCC1855) inoculating 2 weight portions, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of bacillus amyloliquefaciens is 5 × 10
7individual/gram medium.
(3) at medium (glucose 15g, starch 1g, the beancake powder 25g of 100 weight portions, manganese sulphate 1g, potassium dihydrogen phosphate 1.5g, magnesium sulfate 0.5g, yeast extract 0.2g, iron chloride 0.1g, calcium carbonate 0.1g, PH7.0-7.2) the middle husky good fortune bacillus (CCTCCAB205598) inoculating 2 weight portions, 37 DEG C of cultivations, carry out sampling and being observed by ascites method in incubation, until the viable count of husky good fortune bacillus is 5 × 10
7individual/gram medium.
(4) bacillus subtilis (Bacillus subtilis) will obtained respectively in step (1) to (3), , bacillus amyloliquefaciens (Bacillus amyloliquefaciens), husky good fortune bacillus (Bacillus safensis) proportionally mixes, make in the microbial bacterial agent obtained, the viable count of bacillus subtilis (Bacillus subtilis) is 40% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is 30% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is 30% of total viable count, obtain microbial bacterial agent A3.
Embodiment 4
Microbial bacterial agent A4 is prepared according to the method identical with embodiment 3, difference is, by the bacillus subtilis (Bacillus subtilis) obtained respectively in step (1) to (4), , bacillus amyloliquefaciens (Bacillus amyloliquefaciens), husky good fortune bacillus (Bacillus safensis) proportionally mixes, make in the microbial bacterial agent obtained, the viable count of bacillus subtilis (Bacillus subtilis) is 50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is 25% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is 25% of total viable count, obtain microbial bacterial agent A4.
Embodiment 5-8
Microbial bacterial agent A1-A4 obtained in embodiment 1-4 is used to carry out grow seedlings of vegetable respectively, concrete steps are as follows: 2. (1) test material 1. trial crops watermelon, cucumber, tomato, Aztec marigold tests the preparation of microbial nutrition base: microbial bacterial agent A1-A4 is admixed in Nutrient medium raw material according to formula rate mix respectively, then insert in Nutrient medium hydraulic test, be pressed into integrated grow seedling nutritional base.(2) test process.5 process (comprising 1 contrast) are established in test, and each process repeats for 3 times, each repetition 50 strain.Survey item: seedling stage (seedling vigorous index, emergence rate, the incidence of disease), (flowering stage, output, quality, the incidence of disease) (3) test method after field planting.The microbial nutrition base random district group arrangement prepared, put in seedbed, expansion of watering, is sown into vernalization seed in Nutrient medium, covers overburden soil.(4) interpretation of result.Result is as shown in table 1 below.
Comparative example 1
Carry out grow seedlings of vegetable according to the method identical with embodiment 5-8, difference is not use microbial bacterial agent in seedling raising process, and result is as shown in table 1 below.
The statistical analysis of table 1 watermelon indices
As can be seen from the result in upper table 1, microbial bacterial agent provided by the invention has obvious effect to plant indices after watermelon seedling culturing and field planting.As can be seen from Table 1, emergence rate respectively processed and did not have notable difference with contrasting seedling stage; But seedling vigorous index A3 is up to 0.325, contrasting the poorest is 0.077, each process with contrast between reach difference pole significance level, between respectively processing, A2, A3, A4 and A1 reach significance level, do not have difference between A2, A3, A4; Incidence of disease contrast is up to 5%, each process not morbidity; After watermelon seedlings field planting to land for growing field crops, flowering stage is A2 the earliest, and being August 1, is secondly A1, A2, and being contrast the latest, is August 10, illustrates that using microbial bacterial agent nursery to improve yields positive results; Gross yield A3 is up to 1441 kilograms, and minimum is contrast, 1092 kilograms, and each process reaches significance level with contrasting, and does not have difference between process; Quality of watermelon, pol is up to A3 and A4, and minimum is contrast; Watermelon late onset rate is up to contrast, reaches 13.33%, and minimum is A3 and A4, and morbidity Major Diseases is fusarium wilt; Comprehensively use microbial bacterial agent of the present invention can obtain good stand seedling vigorous index as can be seen from above, thus make the aspects such as final yield and quality all obviously be better than contrast, have broad application prospects in grow seedlings of vegetable field.In addition, owing to using microbial bacterial agent provided by the invention can improve plant rhizospheric microorganism environment, beneficial bacterium increases thus suppresses the growth of harmful bacteria, thus can also improve the disease resistance of plant.
Claims (9)
1. a microbial bacterial agent, this microbial bacterial agent comprises medium and thalline, it is characterized in that, described thalline comprises bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and husky good fortune bacillus (Bacillus safensis).
2. microbial bacterial agent according to claim 1, wherein, the total viable count contained by every gram of described microbial bacterial agent is 2-10 × 10
7individual.
3. microbial bacterial agent according to claim 2, wherein, with the total viable count in described microbial bacterial agent for benchmark, the viable count of bacillus subtilis (Bacillus subtilis) is the 10-50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is the 10-50% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is the 10-50% of total viable count.
4. microbial bacterial agent according to claim 3, wherein, with the total viable count in described microbial bacterial agent for benchmark, the viable count of bacillus subtilis (Bacillus subtilis) is the 40-50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is the 15-35% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is the 15-35% of total viable count.
5. the preparation method of microbial bacterial agent according to claim 1, it is characterized in that, the method comprises: bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and husky good fortune bacillus (Bacillus safensis) are inoculated in medium and are cultivated.
6. preparation method according to claim 5, wherein, the described method of carrying out cultivating that is inoculated in medium comprises: in respective independently cultivating system, cultivating bacillus subtilis (Bacillus subtilis), bacillus amyloliquefaciens (Bacillus amyloliquefaciens) and husky good fortune bacillus (Bacillus safensis) respectively, and proportionally mixing cultivating the microorganism obtained respectively.
7. preparation method according to claim 6, wherein, by the cultivation in respective independently cultivating system and the mixing after cultivating, makes total viable count of the every gram of microbial bacterial agent obtained be 2-10 × 10
7individual, wherein, the viable count of bacillus subtilis (Bacillus subtilis) is the 10-50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is the 10-50% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is the 10-50% of total viable count.
8. preparation method according to claim 7, wherein, with the total viable count in described microbial bacterial agent for benchmark, the viable count of bacillus subtilis (Bacillus subtilis) is the 40-50% of total viable count, the viable count of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) is the 15-35% of total viable count, the viable count of husky good fortune bacillus (Bacillus safensis) is the 15-35% of total viable count.
9. the application of the microbial bacterial agent described in any one in claim 1-4 in grow seedlings of vegetable.
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CN105053014A (en) * | 2015-07-06 | 2015-11-18 | 聊城大学 | Composite anti-biological inoculant containing alga matrix, and preparation method and use thereof |
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CN105993461B (en) * | 2016-05-20 | 2021-12-31 | 甘肃省科学院生物研究所 | Seedling raising method of angelica sinensis microbial agent and prevention and treatment method of soil-borne diseases |
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WO2022245786A1 (en) * | 2021-05-18 | 2022-11-24 | AgBiome, Inc. | Compositions and methods for improving plant health and controlling plant disease |
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