CN103215209B - Bacillus amyloliquefaciens and application thereof - Google Patents
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Abstract
The invention discloses bacillus amyloliquefaciens and application thereof, and belongs to the field of biological control of plant diseases. The strain is bacillus amyloliquefaciens RCD-1, and the collection number is CGMCC No.7502. The invention further discloses a biological control preparation containing the strain and a preparation method of the biological control preparation. The bacillus amyloliquefaciens strain provided by the invention has the characteristics of high growth speed, high spore yield, wide effect spectrum and strong stress resistance, and can be fast colonized in quantity at the plant roots, so good application prospect is achieved. The biological control preparation prepared from the bacillus amyloliquefaciens not only can effectively control the soil-borne disease of the plant, but also can increase the crop output, and is a biological control preparation with great application prospect. The microbial preparation can serve as biopesticide or biological fertilizer for controlling various soil-borne plant diseases, especially grape white rot.
Description
Technical field
The invention belongs to biological control Plant diseases field.Specifically, the present invention relates to a strain can the bacillus amyloliquefaciens and uses thereof of high-efficiency prevention and control Plant diseases, and the biological control agent adopting this bacillus amyloliquefaciens to prepare.
Background technology
Grape is defoliation liana, has the title of " god of fruit ", is one of the most ancient in the world plant.Grape originates in Europe, West Asia and north African one band.Be the ground such as Xinjiang, Gansu, Shanxi, Hebei, Shandong in the main place of production of China.Grape both directly can eat as fruit, also can as making grape wine.Grape is edible to be had and antivirally kills the effects such as bacterium, cancer-resisting, reduction hydrochloric acid in gastric juice cholagogic, atherosclerosis, help and excited cerebral nerve, inducing diuresis to remove edema be antiabortive; The grape wine brewageed can invigorating qi and benefiting blood, Yin-nourishing and body fluid promoting, strengthening the muscles and bones, tonneau urine.Cure mainly deficient qi and blood, chronic cough of deficiency lung, the hepatic and renal YIN deficiency, palpitation and night sweat, waist and leg ache, muscles and bones is unable, rheumatic arthralgia, face limb edema, the illnesss such as dysuria.Therefore the grape market requirement is vigorous.But due to the harm of grape disease, have a strong impact on the sound development of grape industry.Fruit white rot of grape (Coniella diplodiella) is that one of heavier disease is endangered in viny region, and the production loss caused by this disease every year reaches 10% ~ 30%, and serious plot reaches more than 50%.This disease pathogen bacterium mainly infects fruit, young stem and inflorescence etc., wherein serious with fruit harm, causes fruit rot and shattering.Disease is once spread, comparatively large to output, quality, Efficiency, causes the underproduction of many orchards even to face the danger in the garden of ruining.For the control of this disease, mainly with bagging or chemical prevention, but often bring the problems such as human cost is too high, medication blindness, super concentration medication.Therefore the biological prevention based on bio-pharmaceutical is the inexorable trend of fruit white rot of grape control.
Genus bacillus is a kind of saprophytic bacteria be distributed widely in various different living environment, is the general name of the product gemma Gram-positive bacillus of the aerobic double anaerobism of a group, can produces the gemma that interior raw heat-resistant is inverse.Gemma not only has resistance to heat, and also have very strong resistance to ultraviolet, electromagnetic radiation and some pharmaceutical chemicalss, it can stand various poor environment.Owing to having so strong resistance, make it be conducive to production and the formulation of biocontrol fungicide, be also beneficial to its survival in the environment, surely grow and breeding.Because their growths are fast, nutrition simple, can form the gemma with stronger resistance ability, the advantage such as high, the stable performance of more non-genus bacillus living bacteria count amount in product development and getting most of the attention.Genus bacillus is the important component part of Plant diseases Biocontrol microorganism, can be used for preventing and treating plurality of plant diseases.Genus bacillus mainly contains as the mechanism of biocontrol microorganisms controlling plant diseases: antibiosis, nutrient competition, competition for space, hyperparasitism and inducing plant produce the mechanism such as resistance, and the Biocontrol Mechanism of genus bacillus mainly suppresses the growth of harmful levels of pathogens to produce antagonistic substance or kills pathogenic bacteria.These materials overwhelming majority is peptide antibiotics, has efficient, low residue and advantages of environment protection.
Summary of the invention
The object of this invention is to provide a kind of new genus bacillus for high-efficiency prevention and control plurality of plant diseases---bacillus amyloliquefaciens RCD-1.This bacterial strain has fast growth, sporulation quantity is large, action spectrum is wide, strong stress resistance, the feature such as can surely to grow by rapid, high volume, therefore have a good application prospect at plant rhizosphere.
Bacterial strain provided by the present invention is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) RCD-1, be separated to obtain from the soil on backlands district, the Weihe River, Weifang City of Shandong Province Coastal beach ground, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 22nd, 2013 and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preserving number is CGMCC No.7502.It has following biological characteristics: on beef extract-peptone nutrient agar or LB substratum, colonial morphology is circular or irregular shape, milky white partially yellow, surface irregularity plays fold, on beef extract-peptone nutrient agar, cultivate two days later at 28 DEG C, microscopy, somatic cells is shaft-like, can move.(taking intestinal bacteria as contrast) is positive through gramstaining.Gemma ovalize or column is observed after spore staining.
Cultural method or the propagation method of bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) bacterial strain RCD-1 comprise:
(1) Nostoc commune Vanch is preserved and is adopted beef extract-peptone agar (NA) substratum, and fill a prescription as Tryptones 10g, extractum carnis 3g, sodium-chlor 5g, agar 15-20g, distilled water 1000mL, pH is adjusted to 7.0-7.2;
(2) laboratory fluids is cultivated and is adopted beef extract-peptone liquid nutrient medium, and fill a prescription as Tryptones 10g, extractum carnis 3g, sodium-chlor 5g, distilled water 1000mL, pH is adjusted to 7.0-7.2;
(3) bulk fermentation cultivates formula: analysis for soybean powder 2.5%, Semen Maydis powder 3.5%, NaCl0.1%, CaCO
30.2%, all the other are water.
The present invention also provides a kind of biological control agent for preventing and treating plant soil-borne diseases, and described biological control agent comprises described Bacillus amyloliquefaciens strain RCD-1.
Further, present invention also offers a kind of biological control method for preventing and treating plant soil-borne diseases, described biological control method comprises uses above-mentioned Bacillus amyloliquefaciens strain RCD-1 or above-mentioned biological control agent to the plant with soil-borne disease.
Present invention provides above-mentioned bacillus amyloliquefaciens bacterium or the purposes of above-mentioned biological control agent in control plant soil-borne diseases.
With regard to above-mentioned biological control agent, biological control method and purposes, described Plant diseases can be selected from comprise fruit white rot of grape, cucumber anthracnose, walnut anthrax, apple anthracnose, bitter rot or anthracnose of grape, watermelon anthrax, winter jujube black spot, Cure of Coryneum kunzei, pomegranate dry rot etc. one or more.
In a specific embodiments of the present invention, described preparation method comprises the following steps:
(1) be transplanted in beef extract-peptone liquid nutrient medium by the spore of Bacillus amyloliquefaciens strain RCD-1,28-30 DEG C of shaking table shaking culture 2-3d obtains seed liquor;
(2) in 3.0% ratio (volume ratio), seed liquor is inoculated in bulk fermentation substratum, culture temperature 28-30 DEG C, incubation time 30h, ventilating ratio 1:0.9.Obtain fermented liquid after having fermented, fermented liquid bacterium number can reach 10,000,000,000/g.
(3) bacillus amyloliquefaciens fermented liquid and matrix (be selected from the peat composed of rotten mosses, micro mist calcium carbonate and wilkinite one or more) are mixed, dry, bacterium number can reach 50,000,000,000/g after drying.The mass ratio of described fermented liquid and fermented liquid matrix is 1:1.
Experiment shows, Bacillus amyloliquefaciens strain of the present invention has fast growth, produce that gemma amount is large, action spectrum wide, strong stress resistance, the feature such as can surely to grow by rapid, high volume, therefore have a good application prospect at plant rhizosphere.The biological control agent prepared by this bacillus amyloliquefaciens, can not only prevent and treat plant soil-borne diseases efficiently, effectively can also improve crop yield, is a kind of biological control agent having application prospect.This microbial preparation can as biological pesticide or bio-feritlizer, prevent and treat multiple soilborne fungal pathogens, comprise fruit white rot of grape, cucumber anthracnose, walnut anthrax, apple anthracnose, bitter rot or anthracnose of grape, watermelon anthrax, winter jujube black spot, Cure of Coryneum kunzei, pomegranate dry rot etc. one or more.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1
1, the isolation and purification of bacillus amyloliquefaciens (B.amyloliquefaciens) bacterial strain RCD-1
Bacillus amyloliquefaciens of the present invention (B.amyloliquefaciens) RCD-1 adopts dilution-plate method to be separated with plate streak and obtains from soil, and separation method is:
(1) separation of genus bacillus: the collection of soil sample, on backlands district, the Weihe River, Weifang City of Shandong Province Coastal beach ground, adopts 5 point samplings, and in right amount, equivalent mixes for collection plot surrounding and the soil of central depth level within the scope of 10-20cm respectively.Indicate collecting location, time and collection people.Take 1g soil sample in 100mL sterilized water, be placed in 30 DEG C of shaking table 150rpm and shake 10min, be then placed in 60 DEG C of water-baths and hatch 30min, get 100 μ L10
-2, 10
-3, 10
-4diluent is coated on beef-protein medium flat board, each gradient be coated with three parallel, the microbial bacteria of the different shape after cultivating 2d at 30 DEG C on picking beef-protein medium falls within beef-protein medium flat board rules, and colony growth situation is observed in timing.Then plate streak is adopted, purifying Bacillus strain, numbering preservation respectively.
(2) the screening of the efficient antagonistic Bacillus of fruit white rot of grape
1. primary dcreening operation: adopt opposite culture method, preparation PDA is dull and stereotyped, buys the bacterium cake that cut-off footpath is 5mm with punch tool at fruit white rot of grape bacterium mycelia edge, is implanted in dull and stereotyped central authorities, and the strain of surrounding 2cm place inoculation genus bacillus, puts 27 DEG C of biochemical cultivation cases and cultivate.Day by day genus bacillus is observed to the restraining effect of pathogenic bacteria.
2. sieve again: the Bacillus strain with efficient antagonistic activity screened is carried out multiple sieve, mainly through temperature tolerance, resistance to acids and bases, drug-resistant test, screens the good Bacillus strain of patience, carry out potted plant controlling experiment and field test.
The present inventor obtains a strain by a large amount of screening operation can bacillus amyloliquefaciens (B.amyloliquefaciens) the bacterial strain RCD-1 of high-efficiency prevention and control plurality of plant diseases.Experiment proves, this bacillus amyloliquefaciens demonstrates prevention effect very efficiently preventing and treating fruit white rot of grape, and farm crop are significantly increased production.Thus, bacillus amyloliquefaciens of the present invention is the bacillus amyloliquefaciens new strains with wide application prospect, may be used for preparing the biological control agent preventing and treating plurality of plant diseases.
2, identification of strains
(1) Microbiological Characteristics: colonial morphology is circular or irregular shape on beef extract-peptone nutrient agar or LB substratum, milky white partially yellow, surface irregularity plays fold, on beef extract-peptone nutrient agar, cultivate at 28 DEG C two days later, microscopy, somatic cells is shaft-like, can move.(taking intestinal bacteria as contrast) is positive through gramstaining.Gemma ovalize or column is observed after spore staining.
(2) molecular biological characteristic
? 16s rDNA of the strain measurement result of gene sequence as follows (SEQ-CGCCTGCCTATACTGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTC?GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGAGCCAGCCGCCGAAGGTG
Embodiment 2
1, Bacillus amyloliquefaciens strain RCD-1 fermenting process
Beef extract-peptone liquid nutrient medium, filling a prescription is: Tryptones 10g, extractum carnis 3g, sodium-chlor 5g, and distilled water 1000mL, pH is adjusted to 7.0-7.2.
A large amount of liquid fermentation medium formula (mass percentage): analysis for soybean powder 2.5%, Semen Maydis powder 3.5%, NaCl0.1%, CaCO
30.2%, all the other are water.
The a large amount of solid fermentation process of Bacillus amyloliquefaciens strain RCD-1:
(1) be transplanted in beef extract-peptone liquid nutrient medium by the spore of Bacillus amyloliquefaciens strain RCD-1,28-30 DEG C of shaking table shaking culture 2-3d obtains seed liquor;
(2) in 3.0% ratio (volume ratio), seed liquor is inoculated in bulk fermentation substratum, culture temperature 28-30 DEG C, incubation time 30h, ventilating ratio 1:0.9.Obtain fermented liquid after having fermented, fermented liquid bacterium number can reach 10,000,000,000/g.
(3) bacillus amyloliquefaciens fermented liquid and matrix (be selected from the peat composed of rotten mosses, micro mist calcium carbonate and wilkinite one or more) are mixed, dry, fermented liquid bacterium number can reach 50,000,000,000/g after drying.The mass ratio of described fermented liquid and fermented liquid matrix is 1:1.
Embodiment 3 Bacillus amyloliquefaciens strain RCD-1 verifies fruit white rot of grape bacterium room effect
1. experimental technique
Adopt dull and stereotyped opposite culture method, fruit white rot of grape bacterium bacterium cake (9mm) for examination is moved to dull and stereotyped central authorities, surrounding 2cm place inoculation bacillus amyloliquefaciens RCD-1 bacterial strain, puts 27 DEG C of biochemical cultivation cases and cultivates, each process repetition 3 times.In time contrasting mycelia and cover with flat board, right-angled intersection method is adopted to measure colony diameter, antibacterial band diameter, calculating mean value and relative inhibition.
2. result
As can be seen from Table 1, Bacillus amyloliquefaciens strain RCD-1 all has stronger restraining effect to fruit white rot of grape bacterium mycelial growth, and antibacterial band diameter is 15.16mm, and mycelial growth inhibition rate is 83.17%.
Table 1. Bacillus amyloliquefaciens strain RCD-1 is to the determination of activity of fruit white rot of grape bacterium Indoor antibacterial
Embodiment 4 Bacillus amyloliquefaciens strain RCD-1 wettable powder is verified fruit white rot of grape field efficacy
Present embodiments provide the related experiment of Bacillus amyloliquefaciens strain RCD-1 wettable powder for fruit white rot of grape.
1.1 reagent agent
Bacillus amyloliquefaciens RCD-1 wettable powder; 50% thiram wettable powder (commercially available).
Bacillus amyloliquefaciens RCD-1 wettable powder agent prescription (weight ratio): the bacterium powder 2%, CMC0.5% of embodiment 2, pulls open powder 3%, sodium laurylsulfonate 8%, glucose 2%, white carbon black 1%, and all the other are attapulgite.The bacterium work of this wettable powder is 1,000,000,000/gram.
1.2 for studying thing and controlling object:
Supplying to study thing is grape, and kind is cigarette 73
Controlling object: white rot
1.3 experimental field situations
Experimental field be located in wine-growing garden, Yantai City, carried out the test of Stand control fruit white rot of grape respectively at 2011 in wine-growing garden, Yantai City.Soil is sand ground.Grapevine is life in 5 years, cultivates from posture.Tree vigo(u)r is medium on the weak side, and disease occurs serious.All experimental plots cultivation condition and control measures consistent
1.4 test design and arrangement
This test design bacillus amyloliquefaciens (B.amyloliquefaciens) RCD-1 wettable powder 300 times, 500 times, 700 times, 50% thiram water dispersible granules 700 times liquid and not dispenser clear water compare totally 5 process, each community 24m
2, repeat 4 times, Gong20Ge community, each community random alignment.June 20 (premorbid) in 2011, later every dispenser in two weeks 1 time, totally 6 times.
1.5 pilot surveies and method of calculation
1.5.1 efficacy survey, control time and number of times
Before fruit harvesting, investigation 1 drug effect, investigates all fruit ears in each community, and record total fruit number, sick fruit number, calculate prevention effect with diseased fruit rate, carry out data analysis with DPS software.
1.5.2 drug effect method of calculation
Drug effect calculates by formula (1), (2):
2. result
2.1 reagent agents are to the prevention effect of fruit white rot of grape
Table 2 result shows, bacillus amyloliquefaciens (B.amyloliquefaciens) RCD-1 wettable powder has good prevention effect to fruit white rot of grape, wherein bacillus amyloliquefaciens (B.amyloliquefaciens) RCD-1 wettable powder 300 times, 500 times prevention effect are best, prevention effect reaches 80.47%, 77.79% respectively, significant difference in P<0.05 level, apparently higher than the prevention effect of 700 times and 50% thiram.
Fruit white rot of grape field control effectiveness test result prevented and treated by table 2 bacillus amyloliquefaciens RCD-1 wettable powder
Note: lowercases different after data in table represents significant difference (P<0.05) after Duncan multiple comparisons.
3. brief summary
From grape diseased fruit rate and prevention effect, 1000000000/gram bacillus amyloliquefaciens (B.amyloliquefaciens) RCD-1 wettable powder to fruit white rot of grape, there is good prevention effect, after diluting 300 times, 500 times, prevention effect can reach more than 77.79%, significantly better than contrast medicament, significant difference.
Claims (5)
1. bacillus amyloliquefaciens (
bacillus amyloliquefaciens) bacterial strain RCD-1, the deposit number of described bacterial strain is CGMCC No.7502.
2. bacillus amyloliquefaciens according to claim 1 (
bacillus amyloliquefaciens) bacterial strain RCD-1 preventing and treating the purposes in fruit white rot of grape.
3. a biological control agent, comprise bacillus amyloliquefaciens according to claim 1 (
bacillus amyloliquefaciens) bacterial strain RCD-1.
4. prepare a method for biological control agent according to claim 3, it is characterized in that, comprise the following steps:
(1) be transplanted in beef extract-peptone liquid nutrient medium by the spore of Bacillus amyloliquefaciens strain RCD-1,28-30 DEG C of shaking table shaking culture 2-3 d obtains seed liquor; Described beef extract-peptone liquid nutrient medium is: Tryptones 10 g, extractum carnis 3 g, sodium-chlor 5 g, distilled water 1000 mL, and pH is adjusted to 7.0-7.2;
(2) seed liquor of step (1) is inoculated in bulk fermentation substratum by the ratio of 3.0% by volume, culture temperature 28-30 DEG C, incubation time 30h, ventilating ratio 1:0.9; Fermented liquid is obtained after having fermented; Described bulk fermentation substratum is: analysis for soybean powder 2.5%, Semen Maydis powder 3.5%, NaCl 0.1%, CaCO
30.2%, all the other are water;
(3) fermented liquid of step (2) is mixed with matrix, dry; The mass ratio of described fermented liquid and matrix is 1:1; Described matrix be selected from the peat composed of rotten mosses, micro mist calcium carbonate and wilkinite one or more.
5. biological control agent according to claim 3 is preventing and treating the purposes in fruit white rot of grape.
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| CN112359001B (en) * | 2020-11-27 | 2022-11-29 | 巴州加木农业科技有限公司 | Bacillus amyloliquefaciens microbial agent and application thereof |
| CN113980859B (en) * | 2021-11-22 | 2023-03-31 | 中国科学院微生物研究所 | Bacillus amyloliquefaciens and application thereof |
| CN117431194B (en) * | 2023-12-21 | 2024-03-19 | 山东省林业科学研究院 | Bacillus amyloliquefaciens, composition and application thereof in cut-flower Paeonia lactiflora |
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