CN103215209A - 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.Particularly, the present invention relates to bacillus amyloliquefaciens that a strain can the high-efficiency prevention and control Plant diseases and uses thereof, and the biological control preparation that adopts this bacillus amyloliquefaciens preparation.
Background technology
Grape is a defoliation liana, and the title of " god of fruit " is arranged, and is one of the most ancient in the world plant.Grape originates in Europe, West Asia and north African one band.In the main place of production of China is ground such as Xinjiang, Gansu, Shanxi, Hebei, Shandong.It is directly edible that grape both can be used as fruit, also can be as making grape wine.Grape is edible to have the antiviral 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 of killing; But the grape wine invigorating qi and benefiting blood of brewageing, 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, the waist and leg ache, muscles and bones is unable, rheumatic arthralgia, face limb edema, illnesss such as dysuria.Therefore the grape market requirement is vigorous.But because the harm of grape disease has had a strong impact on the sound development of grape industry.Fruit white rot of grape (Coniella diplodiella) is one of heavier disease of viny region harm, and the annual production loss that is caused by this disease reaches 10%～30%, and serious plot reaches more than 50%.This disease pathogen bacterium is mainly infected fruit, young stem and inflorescence etc., and is wherein serious with fruit harm, causes fruit rot and shattering.In a single day disease spreads, bigger to output, quality, benefit influence, causes the underproduction of many orchards even faces the danger in the garden of ruining.At the control of this disease,, but tend to bring problems such as human cost is too high, medication blindness, super concentration medication mainly with bagging or chemical prevention.Therefore the biological control technology based on bio-pharmaceutical is the inexorable trend of fruit white rot of grape control.
Genus bacillus is a kind of saprophytic bacteria that is distributed widely in the various different living environments, is the general name that the aerobic double anaerobic of a group produces the gemma Gram-positive bacillus, gives birth to heat-resisting degeneration-resistant gemma in can producing.Gemma not only has resistance to heat, also ultraviolet ray, electromagnetic radiation and some pharmaceutical chemicalss is had very strong resistance, and it can stand various poor environments.Owing to have so strong resistance, make it help the production and the formulation processing of biocontrol fungicide, also be beneficial to its survival in environment, grow and breeding surely.Because their growths are fast, nutrition simple, can form the gemma with strong resistance ability, advantage such as more non-genus bacillus living bacteria count amount height, stable performance in product development and getting most of the attention.Genus bacillus is the important component part of Plant diseases biological and ecological methods to prevent plant disease, pests, and erosion 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 mechanism such as resistance, and the Biocontrol Mechanism of genus bacillus mainly is to suppress harmful pathogen growth or kill pathogenic bacteria to produce antagonistic substance.These materials overwhelming majority is a peptide antibiotics, has efficient, low residue and advantages of environment protection.
Summary of the invention
The purpose of this invention is to provide a kind of new genus bacillus that is used for the high-efficiency prevention and control plurality of plant diseases---bacillus amyloliquefaciens RCD-1.This bacterial strain has fast growth, sporulation quantity is big, action spectrum is wide, strong stress resistance, characteristics such as can grow fast in a large number surely at plant rhizosphere, therefore has a good application prospect.
Bacterial strain provided by the present invention is bacillus amyloliquefaciens (Bacillus amyloliquefaciens) RCD-1, be from the soil on coastal beach ground, backlands, the Weihe River, Weifang City, Shandong Province district, to separate to obtain, be preserved in China Committee for Culture Collection of Microorganisms 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: colonial morphology is circular or irregular shape on beef extract-peptone nutrient agar or LB substratum, milky white yellow partially, surface irregularity plays fold, on the beef extract-peptone nutrient agar, cultivate two days later down for 28 ℃, microscopy, somatic cells are shaft-like, can move.Through gramstaining be positive (being contrast with intestinal bacteria).After spore staining, observe gemma ovalize or column.
Cultural method or the propagation method of bacillus amyloliquefaciens of the present invention (Bacillus amyloliquefaciens) bacterial strain RCD-1 comprise:
(1) common cultivation is preserved and is adopted beef extract-peptone agar (NA) substratum, and filling a prescription is Tryptones 10g, extractum carnis 3g, and sodium-chlor 5g, agar 15-20g, distilled water 1000mL, pH transfers to 7.0-7.2;
(2) laboratory fluids is cultivated and is adopted the beef extract-peptone liquid nutrient medium, and filling a prescription is Tryptones 10g, extractum carnis 3g, and sodium-chlor 5g, distilled water 1000mL, pH transfers to 7.0-7.2;
(3) bulk fermentation is cultivated prescription: 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 preparation that is used to prevent and treat plant soil-borne diseases, and described biological control preparation comprises described bacillus amyloliquefaciens bacterial strain RCD-1.
And the present invention also provides a kind of biological control method that is used to prevent and treat plant soil-borne diseases, and described biological control method comprises to the plant with soil-borne disease uses above-mentioned bacillus amyloliquefaciens bacterial strain RCD-1 or above-mentioned biological control preparation.
The present invention also provides above-mentioned bacillus amyloliquefaciens bacterium or the purposes of above-mentioned biological control preparation in the control plant soil-borne diseases.
With regard to above-mentioned biological control preparation, 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 may further comprise the steps:
(1) spore of bacillus amyloliquefaciens bacterial strain RCD-1 is transplanted in the beef extract-peptone liquid nutrient medium, 28-30 ℃ of shaking table shaking culture 2-3d obtains seed liquor;
(2) in 3.0% ratio (volume ratio) seed liquor is inoculated in the bulk fermentation substratum, culture temperature 28-30 ℃, incubation time 30h, ventilating ratio 1:0.9.Obtain fermented liquid after fermentation is finished, 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 lime carbonate and the wilkinite one or more) are mixed, drying, the 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 bacterial strain of the present invention has fast growth, produce that the gemma amount is big, action spectrum wide, strong stress resistance, characteristics such as can grow fast in a large number surely at plant rhizosphere, therefore has a good application prospect.By the biological control preparation of this bacillus amyloliquefaciens preparation, not only can prevent and treat plant soil-borne diseases efficiently, can also effectively improve crop yield, be a kind of biological control preparation that has application prospect.This microbial preparation can be used as biological pesticide or bio-feritlizer, prevent and treat multiple soil and pass Plant diseases, 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 of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
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 separate acquisition with plate streak from soil, and separation method is:
(1) separation of bacillus: the collection of soil sample, on coastal beach ground, backlands, the Weihe River, Weifang City, Shandong Province district, adopt 5 point samplings, gather around the plot respectively and the soil of the central degree of depth in the 10-20cm scope an amount of, the equivalent mixing.Indicate collecting location, time and collection people.Take by weighing the 1g soil sample in the 100mL sterilized water, place 30 ℃ of shaking table 150rpm concussion 10min, place 60 ℃ of water-baths to hatch 30min then, get 100 μ L10
-2, 10
-3, 10
-4Diluent is coated on the beef-protein medium flat board, each three of gradient coating is parallel, the microbial bacteria of the different shape on picking beef-protein medium behind 30 ℃ of cultivation 2d falls within on the beef-protein medium flat board rules, and regularly observes the colony growth situation.Adopt plate streak then, the purifying Bacillus strain, numbering is preserved respectively.
⑵ the screening of the efficient antagonism genus bacillus of fruit white rot of grape
1. primary dcreening operation: adopt the face-off culture method, preparation PDA flat board is bought the bacterium cake that cut-off directly is 5mm with punch tool at fruit white rot of grape bacterium mycelia edge, is implanted in dull and stereotyped central authorities, and 2cm place inoculates the genus bacillus strain all around, puts 27 ℃ of biochemical incubators cultivations.Day by day observe the restraining effect of genus bacillus to pathogenic bacteria.
2. sieve again: the Bacillus strain with efficient antagonistic activity that will screen carries out multiple sieve, mainly is through temperature tolerance, resistance to acids and bases, drug-resistant test, screens patience Bacillus strain preferably, carries out potted plant control test and field test.
The inventor obtains bacillus amyloliquefaciens (B.amyloliquefaciens) the bacterial strain RCD-1 that a strain can the high-efficiency prevention and control plurality of plant diseases by a large amount of screening operations.Experiment showed, that this bacillus amyloliquefaciens demonstrates prevention effect very efficiently preventing and treating fruit white rot of grape, makes farm crop significantly increase production.Thereby bacillus amyloliquefaciens of the present invention is the new bacterial strain of bacillus amyloliquefaciens with wide application prospect, can be used to prepare the biological control preparation of 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, and milky white yellow partially, surface irregularity plays fold, on the beef extract-peptone nutrient agar, cultivate two days later microscopy down for 28 ℃, it is shaft-like that somatic cells is, and can move.Through gramstaining be positive (being contrast with intestinal bacteria).After spore staining, observe gemma ovalize or column.
(2) molecular biological characteristic
The strain 16s? RDNA gene sequence determination results are as follows（SEQ-1）CGCCTGCCTATACTGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTC GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGAGCCAGCCGCCGAAGGTG
Embodiment 2
1, bacillus amyloliquefaciens bacterial strain RCD-1 fermenting process
The beef extract-peptone liquid nutrient medium, filling a prescription is: Tryptones 10g, extractum carnis 3g, sodium-chlor 5g, distilled water 1000mL, pH transfers to 7.0-7.2.
A large amount of liquid fermentation medium prescriptions (quality percentage composition): 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 processes of bacillus amyloliquefaciens bacterial strain RCD-1:
(1) spore of bacillus amyloliquefaciens bacterial strain RCD-1 is transplanted in the beef extract-peptone liquid nutrient medium, 28-30 ℃ of shaking table shaking culture 2-3d obtains seed liquor;
(2) in 3.0% ratio (volume ratio) seed liquor is inoculated in the bulk fermentation substratum, culture temperature 28-30 ℃, incubation time 30h, ventilating ratio 1:0.9.Obtain fermented liquid after fermentation is finished, 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 lime carbonate and the wilkinite one or more) are mixed, drying, 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 bacterial strain RCD-1 verify fruit white rot of grape bacterium room effect
1. experimental technique
Adopt dull and stereotyped face-off culture method, will move to dull and stereotyped central authorities for the fruit white rot of grape bacterium bacterium cake (9mm) of examination, the bacillus amyloliquefaciens of 2cm place inoculation all around RCD-1 bacterial strain is put 27 ℃ of biochemical incubators and is cultivated, and each is handled and repeats 3 times.When mycelia to be contrasted is covered with flat board, adopt the right-angled intersection method to measure colony diameter, antibacterial band diameter, calculating mean value and relative inhibition.
2. result
As can be seen from Table 1, bacillus amyloliquefaciens bacterial 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 bacterial strain RCD-1 measures the indoor bacteriostatic activity of fruit white rot of grape bacterium
Embodiment 4 bacillus amyloliquefaciens bacterial strain RCD-1 wettable powders are to fruit white rot of grape field compliance test result
Present embodiment provides the related experiment of bacillus amyloliquefaciens bacterial strain RCD-1 wettable powder at fruit white rot of grape.
1.1 reagent agent
Bacillus amyloliquefaciens RCD-1 wettable powder; 50% thiram wettable powder (commercially available).
The bacterium powder 2% of bacillus amyloliquefaciens RCD-1 wettable powder agent prescription (weight ratio): embodiment 2, CMC0.5% draws back powder 3%, sodium laurylsulfonate 8%, glucose 2%, white carbon black 1%, 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:
For studying thing is grape, and kind is a cigarette 73
Controlling object: white rot
1.3 situation experimental field
Experimental field be located in the wine-growing garden, Yantai City, Shandong Province, carried out woodland respectively at 2011 in wine-growing garden, Yantai City and prevent and treat the fruit white rot of grape test.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 takes place serious.All experimental plot cultivation conditions and control measures unanimity
1.4 test design and arrangement
This test design 300 times, 500 times, 700 times of bacillus amyloliquefaciens (B.amyloliquefaciens) RCD-1 wettable powders, 700 times of liquid of 50% thiram water dispersible granules and not dispenser clear water compare totally 5 processing, each sub-district 24m
2, repeat totally 20 sub-districts, each sub-district random alignment 4 times.June 20 (premorbid) in 2011, later on every two all dispensers 1 time, totally 6 times.
1.5 pilot survey and method of calculation
1.5.1 efficacy survey, control time and number of times
1 drug effect of investigation is investigated all fruit ears of each sub-district before the fruit harvesting, and the total fruit of record 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 is calculated by formula (1), (2):
2. result
2.1 reagent agent is to the prevention effect of fruit white rot of grape
Table 2 result shows, bacillus amyloliquefaciens (B.amyloliquefaciens) RCD-1 wettable powder has the better prevention effect to fruit white rot of grape, wherein 300 times, 500 times prevention effect of bacillus amyloliquefaciens (B.amyloliquefaciens) RCD-1 wettable powder are best, prevention effect reaches 80.47%, 77.79% respectively, significant difference on P＜0.05 level is apparently higher than the prevention effect of 700 times and 50% thiram.
Table 2 bacillus amyloliquefaciens RCD-1 wettable powder is prevented and treated fruit white rot of grape field control effectiveness test result
Annotate: in the table after the data different lowercases represent significant difference behind the Duncan multiple comparisons (P＜0.05).
3. brief summary
From grape diseased fruit rate and prevention effect, bacillus amyloliquefaciens (B.amyloliquefaciens) the RCD-1 wettable powder of 1,000,000,000/gram has the better prevention effect to fruit white rot of grape, prevention effect can reach more than 77.79% after diluting 300 times, 500 times, significantly better than contrast medicament, significant difference.
Claims (8)
1. bacillus amyloliquefaciens (Bacillus amyloliquefaciens) RCD-1, the deposit number of described bacterial strain is CGMCC No.7502.
2. the described bacillus amyloliquefaciens of claim 1 (Bacillus amyloliquefaciens) bacterial strain RCD-1 is in the purposes of control in the plant soil-borne diseases.
3. purposes as claimed in claim 2, it is characterized in that described plant soil-borne diseases is one or more in 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, the pomegranate dry rot.
4. a biological control preparation comprises the described bacillus amyloliquefaciens of claim 1 (Bacillus amyloliquefaciens) bacterial strain RCD-1.
5. a method for preparing the described biological control preparation of claim 4 is characterized in that, may further comprise the steps:
(1) spore of bacillus amyloliquefaciens bacterial strain RCD-1 is transplanted in the beef extract-peptone liquid nutrient medium, 28-30 ℃ of shaking table shaking culture 2-3d obtains seed liquor; Described beef extract-peptone liquid nutrient medium is: Tryptones 10g, and extractum carnis 3g, sodium-chlor 5g, distilled water 1000mL, pH transfers to 7.0-7.2;
(2) 3.0% ratio is inoculated into the seed liquor of step (1) in the bulk fermentation substratum by volume, and culture temperature 28-30 ℃, incubation time 30h, ventilating ratio 1:0.9; After finishing, fermentation obtains fermented liquid; Described bulk fermentation substratum is: analysis for soybean powder 2.5%, and Semen Maydis powder 3.5%, NaCl 0.1%, CaCO
30.2%, all the other are water;
(3) fermented liquid and the matrix with step (2) mixes drying; The mass ratio of described fermented liquid and matrix is 1:1; Described matrix is selected from one or more in the peat composed of rotten mosses, micro mist lime carbonate and the wilkinite.
6. the described biological control preparation of claim 4 is in the purposes of control in the plant soil-borne diseases.
7. purposes as claimed in claim 6, it is characterized in that described plant soil-borne diseases is one or more in 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, the pomegranate dry rot.
8. the biological control method of a plant soil-borne diseases is characterized in that, uses bacillus amyloliquefaciens (Bacillus amyloliquefaciens) the bacterial strain RCD-1 of claim 1 or the biological control preparation of claim 4 to the plant with soil-borne disease.
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