CN102154155A - Brevibacillus brevis for preventing and treating plant fungus diseases and method for preparing biopesticide - Google Patents
Brevibacillus brevis for preventing and treating plant fungus diseases and method for preparing biopesticide Download PDFInfo
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Abstract
The invention relates to brevibacillus brevis for preventing and treating plant fungous diseases and application thereof. The brevibacillus brevis has a gene sequencing result of 1367bp, has an accession number of HQ585418 on a gene bank, is preserved in the China general microbiological culture collection center (CGMCC) and has a preservation number of CGMCC NO.4361. A method for preparing a brevibacillus brevis biopesticide for preventing and treating the plant fungous diseases comprises the following operation steps of: 1, preparing seed liquid; and 2, fermenting and preparing the brevibacillus brevis biopesticide. A field experiment proves that the brevibacillus brevis can obviously reduce the morbidity of cucurbit wilt, and thus, a strain disclosed by the invention can be effectively applied to prevention and treatment of cucurbit wilt and can be used for preparing the safe and high-efficiency biopesticide.
Description
Technical field
The invention belongs to biological pesticide technical field, be specifically related to prevent and treat the short genus bacillus of fungal diseases of plants and the preparation method of biological pesticide thereof.
Background technology
The life-time service of chemical pesticide has caused serious consequence: the pesticide residue in agricultural-food and the soil is more and more higher, and agricultural product quality is seriously descended; The density of beneficial organism descends, and natural community has suffered havoc; The resistance of pathogenic bacteria and insect generally improves, and causes the excess of chemical pesticide to be used, and has improved the production cost of agricultural-food, and has aggravated environment damage has been formed vicious cycle.Along with the enhancing of people's environmental consciousness, plurality of advantages such as biological pesticide is free from environmental pollution because of it,, pathogenic bacteria strong to people and animals and plants safety, selectivity and insect are difficult to produce resistance is subjected to the great attention of various countries, is the main direction of agricultural chemicals development from now on.At present, in the applications of pesticide of China, biological pesticide only accounts for 1% ratio, and price is low, kind is few.Plant diseases is one of natural disaster that threatens farm crop production, Plant diseases above 80% in the production is caused by fungal infection, when this disease seriously takes place, can cause the farm crop serious decline of the underproduction and quality significantly, even cause the person poultry poisoning, have a strong impact on the national economy and the people's life.Therefore, the biological pesticide of seeking antifungal plant disease of new generation under the background of considering environment and food safety seems very necessary and urgent.
Blight is a kind of common important disease on melon, the cotton.The pathogenic bacteria of blight be the imperfect fungi Fusarium oxysporum (
Fusarium oxysporum.f.sp), this germ host range is extremely wide, generally causes the crop root browning to rot, and the basal part of stem lobe that contracts of hanging can be drawn various crop such as watermelon, tomato, cotton destructive disease is taken place, and produces to farm crop and causes heavy losses.This germ mainly is present in the soil with mycelia, chlamydospore or sclerotium, can survive 6～10 years, and can be by seed, fertilizer, soil, the propagation of watering, refractory is difficult anti-, is called as " melon cancer ", " cotton cancer " etc.Aspect the preventing and treating of farm crop blight, because intractable, diversity and the variability of pathogenic bacteria, still lack effective chemical medicament and effective disease-resistant variety at present, become and be badly in need of the key issue that solves on the crop production such as melon.The various countries scholar wishes to seek the breach in the biological control of " controlling bacterium with bacterium ", utilizes biological pesticide to prevent and treat blight, and is carrying out positive experimental study for this reason.
Summary of the invention
The purpose of this invention is to provide a kind of can be in the plant rhizosphere definite value, and the short genus bacillus of the biocontrol microorganisms that multiple pathogenic bacteria is had antagonistic action----.
Another object of the present invention provides a kind of preparation method who prevents and treats the short genus bacillus biological pesticide of fungal diseases of plants.
The 16S rDNA gene order of the short Bacillus strain of control fungal diseases of plants is as follows:
CTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTCTCAGACTGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGTTTTTGGATCGCATGATCCGAAAAGAAAAGATGGCTTCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTTAGGGACGAATAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGGGCTCAACTCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGGTTTCAATACCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGCTCTGGAGACAGAGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGGGATGCTACCTCGCGAGAGGACGCCAATCTCTTAAAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCGCAAGG，
Sequencing result is:
1367bp;The accession number of the gene pool (GeneBank) of the U.S. state-run biotechnology center website is HQ585418.
The screening method of the short genus bacillus of control fungal diseases of plants comprises following operation steps:
(1) soil sampling: the watermelon field that selection is grown fine, at the rhizosphere of healthy watermelon plant, scalp topsoil 2-3cm, get the soil 10g that closes on the watermelon root; Pack into and went out in the kraft bag of bacterium, seal sack, and record sampling spot, environment and the date.
(2) strains separation and purifying: the soil sample of fetching is adopted gradient dilution plate streak separation and purification bacterial strain wherein, and concrete operations are as follows:
With ready solid medium (NA substratum) heating and melting, preparation is dull and stereotyped; Take by weighing soil sample 1g, add in the 10ml sterilized water, and add sterilization granulated glass sphere vibration 15 minutes, determine that concentration is 1, get supernatant liquor 1.0mL after leaving standstill and join in the test tube that fills the 9.0mL sterilized water, vibration shakes up, and concentration is 10
-1, this strength of solution to 10 of gradient dilution successively
-2, 10
-3, 10
-410
-11Get each gradient solution 50uL and join respectively in beef-protein medium (NA) flat board, coating is evenly cultivated in 30 ℃ thermostat container, cultivates 2-7 days; The bacterial strain that the picking form differs, method of scoring purifying 2-3 time of each bacterial strain.
(3) active bacterial strain screening:
Primary dcreening operation: adopt agar to move piece face-off laboratory method, concrete operations are as follows:
Even coating withered germ of water-melon on potato glucose substratum (PDA) flat board (
Fusarium oxysporumF sp.Niveum), 30 ℃ of cultivations treat that pathogenic bacteria grows (3-5 days), beat to get with punch tool to contain the bacterio-agar piece; To contain the central position that the bacterio-agar piece is put into the PDA culture medium flat plate, to the symmetric position from withered germ of water-melon 20mm, each dull and stereotyped going up is inoculated four bacterial strains to be measured with the isolated inoculation to be measured of step 2; Cultivated 3-5 days for 30 ℃, observe bacteriostatic activity,, obtain antagonistic strain according to having or not or the big or small antagonistic activity of determining bacterial strain to be measured of antibacterial band;
Multiple sieve: for determining reliability and its antagonism performance size of quantitative comparison of primary dcreening operation antagonistic strain, the antagonistic strain that obtains in the primary dcreening operation is carried out multiple sieve, concrete operations are as follows;
With primary dcreening operation antagonistic strain NA liquid nutrient medium, shake flask fermentation is cultivated 24h under 30 ℃ of temperature, rotating speed 160r/min condition, the centrifugal thalline that goes, get supernatant, detect the fungistatic effect of fermented liquid supernatant with cylinder plate method, weigh the fungistatic effect of antagonistic strain with the size of antibacterial area, obtain the very strong short Bacillus strain of fungistatic effect, its antibacterial area is 735mm
2
Short Bacillus strain has following morphological specificity and physiological and biochemical property:
Colony morphology characteristic: go up single bacterium colony circle, neat in edge at beef-protein medium (NA), flat, smooth, greyish white slightly yellow, the no soluble pigment in surface combines closely with substratum;
Morphological features: cell is shaft-like, and Gram-positive includes circular gemma expanding sporangiocyst, and the gemma end is given birth to;
Physiological and biochemical property: not hydrolyzed starch, the gelatine liquefication ability is strong, V-P is negative, do not produce hydrogen sulfide and indoles, the M.R test is negative, catalase test is positive, sugar fermentating test is negative, produce the ammonia test feminine gender, can under the acidic conditions of pH5.7, grow, can not under 50 ℃, 7% NaC1 condition, grow;
To the fungi antagonistic ability: the efficient antagonistic effect that on potato glucose (PDA) substratum, shows the pathogenic bacteria to watermelon blight, wooden enzyme etc.
The 16S rDNA sequencing result of short genus bacillus provided by the present invention is delivered to the gene pool (GeneBank) of the U.S. state-run biotechnology center website, accession number is HQ585418, on http://www.ncbi.nlm.nih.gov/ website, compare with blast program and the 16S rDNA gene order of logining bacterial isolates, the result shows that the similarity of this bacterial strain and short genus bacillus is the highest, reach more than 99.5%, sibship is nearest, and with short genus bacillus
Brevibacillus brevis(EF173465) in a branch, see Fig. 1.
The deposit number of short genus bacillus provided by the present invention is CGMCC NO.4361, is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, the depositary institution address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preservation date is on November 25th, 2010, the classification short genus bacillus of called after (Brevibacillus brevis).
The preparation method of the short genus bacillus biological pesticide of control fungal diseases of plants comprises following operation steps:
(1) short genus bacillus is inoculated in the 250ml triangular flask of every bottled 50ml beef extract-peptone (NA) nutrient solution, 29 ℃ of shaking culture 24h, shaking speed 170.00r/min makes seed liquor;
Beef extract-peptone (NA) culture medium prescription is peptone 1.0%, sodium-chlor 0.5%, extractum carnis 0.5%, and initial pH value is 7.2-7.4,121 ℃ of sterilization 20min.
(2) in 5 liters of automatic fermentor tanks, fermention medium tinning 75%, by 10% inoculum size inoculation seed liquor, ventilation 3NL/min cultivates 36h down in 29 ℃, puts jar, and fermented liquid is regulated matter 10 behind the microscopy counting
8-10
10Cell/ml is the biological pesticide that contains short genus bacillus;
Described fermentative medium formula is glucose 1.00%, peptone 2.23%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate heptahydrate 0.05%, and initial pH value is 7.50, real jar of sterilization.
Useful technique effect of the present invention embodies in the following areas:
1, antagonistic strain of the present invention can obviously suppress the watermelon blight growth of pathogenic bacteria, field experiment also can obviously reduce the sickness rate of plant, therefore, bacterial strain fermentation liquor of the present invention can be effectively applied to the control of watermelon blight, and can be used to prepare a kind of safe, biological pesticide efficiently.
2, the short genus bacillus biocontrol agent of being produced by technical solution of the present invention can high-efficiency prevention and control fungi plant disease, and nuisanceless.
Description of drawings
The bacterial strain systematic evolution tree analysis that Fig. 1 relates to for this patent.
Fig. 2 detects the fungistatic effect figure of bacterium to part for short fermentation of bacillus liquid.
Fig. 3 is that root experiment effect figure is irritated in short fermentation of bacillus liquid field.
Embodiment
Below in conjunction with embodiment, the present invention is done to describe further.
Embodiment
The screening method of the short genus bacillus of control fungal diseases of plants comprises following operation steps:
(1) soil sampling: the watermelon field that selection is grown fine, at the rhizosphere of healthy watermelon plant, scalp topsoil 2-3cm, get the soil 10g that closes on the watermelon root; Pack into and went out in the kraft bag of bacterium, seal sack, and record sampling spot, environment and the date.
(2) strains separation and purifying: the soil sample of fetching is adopted gradient dilution plate streak separation and purification bacterial strain wherein, and concrete operations are as follows:
With ready beef extract-peptone solid medium (NA substratum) heating and melting, preparation is dull and stereotyped; Take by weighing soil sample 1g, add in the 10ml sterilized water, and add sterilization granulated glass sphere vibration 15 minutes, determine that concentration is 1, get supernatant liquor 1.0mL after leaving standstill and join in the test tube that fills the 9.0mL sterilized water, vibration shakes up, and concentration is 10
-1, this strength of solution to 10 of gradient dilution successively
-2, 10
-3, 10
-410
-11Get each gradient solution 50uL and join respectively in the NA flat board, coating is evenly cultivated in 30 ℃ thermostat container, cultivates 2-7 days; The bacterial strain that the picking form differs, method of scoring purifying 2-3 time of each bacterial strain;
(3) active bacterial strain screening:
Primary dcreening operation: adopt agar to move piece face-off laboratory method, concrete operations are as follows:
Even coating withered germ of water-melon on potato glucose substratum (PDA) flat board (
Fusarium oxysporumF sp.Niveum), 30 ℃ of cultivations treat that pathogenic bacteria grows (3-5 days), beat to get with punch tool to contain the bacterio-agar piece; To contain the central position that the bacterio-agar piece is put into the PDA culture medium flat plate, to the symmetric position from withered germ of water-melon 20mm, each dull and stereotyped going up is inoculated four bacterial strains to be measured with the isolated inoculation to be measured of step 2; Cultivated 3-5 days for 30 ℃, observe bacteriostatic activity,, obtain antagonistic strain according to having or not or the big or small antagonistic activity of determining bacterial strain to be measured of antibacterial band;
Multiple sieve: for determining reliability and its antagonism performance size of quantitative comparison of primary dcreening operation antagonistic strain, the antagonistic strain that obtains in the primary dcreening operation is carried out multiple sieve, concrete operations are as follows;
With primary dcreening operation antagonistic strain NA liquid nutrient medium, shake flask fermentation is cultivated 24h under 30 ℃ of temperature, rotating speed 160r/min condition, the centrifugal thalline that goes, get supernatant, detect the fungistatic effect of fermented liquid supernatant with cylinder plate method, weigh the fungistatic effect of antagonistic strain with the size of antibacterial area, obtain the very strong short Bacillus strain of fungistatic effect, its antibacterial area is 735mm
2
Short Bacillus strain has following morphological specificity and physiological and biochemical property:
Colony morphology characteristic: go up single bacterium colony circle, neat in edge at beef-protein medium (NA), flat, smooth, greyish white slightly yellow, the no soluble pigment in surface combines closely with substratum;
Morphological features: cell is shaft-like, and Gram-positive includes circular gemma expanding sporangiocyst, and the gemma end is given birth to;
Physiological and biochemical property: not hydrolyzed starch, the gelatine liquefication ability is strong, V-P is negative, do not produce hydrogen sulfide and indoles, the M.R test is negative, catalase test is positive, sugar fermentating test is negative, produce the ammonia test feminine gender, can under the acidic conditions of pH5.7, grow, can not under 50 ℃, 7% NaC1 condition, grow;
With the conservative property universal primer at the 16S rDNA sequence two ends of the bacterium of different genera, to the above-mentioned short genus bacillus 16S rDNA order-checking of increasing:
Front end primer 1 is 5 ' AGAGTTTGATCCTGGCTCAG3 ', and the rear end primer 2 is 5 ' ACGGTTACCTTGTTACGACTT 3 '
The gene order of short genus bacillus that obtains preventing and treating fungal diseases of plants is as follows:
CTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTCTCAGACTGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGTTTTTGGATCGCATGATCCGAAAAGAAAAGATGGCTTCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTTAGGGACGAATAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGGGCTCAACTCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGGTTTCAATACCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGCTCTGGAGACAGAGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGGGATGCTACCTCGCGAGAGGACGCCAATCTCTTAAAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCGCAAGG，
Sequencing result is:
1367bp;The accession number of the gene pool (GeneBank) of the U.S. state-run biotechnology center website is HQ585418.
The preparation method of the short genus bacillus biological pesticide of control fungal diseases of plants comprises following operation steps:
(1) short genus bacillus is inoculated in the 250ml triangular flask of every bottled 50ml beef extract-peptone (NA) nutrient solution, 29 ℃ of shaking culture 24h, shaking speed 170.00r/min makes seed liquor;
Beef extract-peptone (NA) culture medium prescription is peptone 1.0%, sodium-chlor 0.5%, extractum carnis 0.5%, and initial pH value is 7.2-7.4,121 ℃ of sterilization 20min.
(2) in 5 liters of automatic fermentor tanks, fermention medium tinning 75%, by 10% inoculum size inoculation seed liquor, ventilation 3NL/min cultivates 36h down in 29 ℃, puts jar, and fermented liquid is behind the microscopy counting, and regulating concentration is 10
8-10
10Cell/ml, aseptic subpackaged, the short fermentation of bacillus liquid that obtains is the biological pesticide that contains short genus bacillus;
Described fermentative medium formula is glucose 1.00%, peptone 2.23%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate heptahydrate 0.05%, and initial pH value is 7.50, real jar of sterilization.
The activity of the short fermentation of bacillus liquid of the present invention detects
Short fermentation of bacillus liquid has antimicrobial spectrum widely, detects its bacteriostatic activity with cylinder plate method:
1, strains tested source: colon bacillus, streptococcus aureus, pichia yeast bacterium, Leuconostoc mesenteroides, Li's Trichoderma, watermelon blight source bacterium are all by this laboratory preservation.
2, cups and dishes detection method: adopt cup-plate method to measure, add fermented supernatant fluid 300 μ L in the Oxford cup (diameter is 7mm), the size with inhibition zone cartographic represenation of area bacteriostatic activity the results are shown in Table 1; The fungistatic effect of part indicator is seen Fig. 2.
The bacteriostatic activity result of table 1 antimicrobial substance
The field experiment that contains the biological pesticide control watermelon blight of short genus bacillus
The sick spectrum of blight field is positioned at Shuan Dun town, Changfeng County, do the test kind with height sense variety of watermelon honey treasured, water normal root water during transplanting, three concentration of biological pesticide dilution that contain short genus bacillus: 2 times, 4 times, 8 times, clear water is done contrast, every processing 10 strains, three repetitions, 20--50 days observation watermelon plant incidences; Handle high anti-kind with the biological pesticide that contains short genus bacillus of 2 times of dilutions simultaneously, clear water is treated to contrast, observes the watermelon growing way.Experimental result: 2 times and the 4 times sweet precious plant of irritating root of biological pesticide dilution that contain short genus bacillus grow fine, and survival rate is 95%; Diluting 8 times of plant survival rates of irritating root is 80%, and growing way is normal; Clear water is irritated and to be taken root in strain and fall ill substantially or stop growing, and sees Fig. 3.Show that the short genus bacillus of biocontrol microorganisms has very strong antagonistic effect, can reduce the harm of blight to the watermelon plant greatly.Growing way obviously was better than the clear water processing after disease-resistant variety of watermelon diluted 2 times of processing with the biological pesticide that contains short genus bacillus, showed the effect that promotes the watermelon plant strain growth.
Claims (2)
1. prevent and treat the short genus bacillus of fungal diseases of plants, it is characterized in that: the 16S rDNA gene order of described short Bacillus strain is as follows:
CTAGCGGCGGACGGGTGAGTAACACGTAGGCAACCTGCCTCTCAGACTGGGATAACATAGGGAAACTTATGCTAATACCGGATAGGTTTTTGGATCGCATGATCCGAAAAGAAAAGATGGCTTCGGCTATCACTGGGAGATGGGCCTGCGGCGCATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATTTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAACGATGAAGGTCTTCGGATTGTAAAGTTCTGTTGTTAGGGACGAATAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTGACGAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGCGCGCAGGCGGCTATGTAAGTCTGGTGTTAAAGCCCGGGGCTCAACTCCGGTTCGCATCGGAAACTGTGTAGCTTGAGTGCAGAAGAGGAAAGCGGTATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTGGGGGTTTCAATACCCTCAGTGCCGCAGCTAACGCAATAAGCACTCCGCCTGGGGAGTACGCTCGCAAGAGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCGCTGACCGCTCTGGAGACAGAGCTTCCCTTCGGGGCAGCGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTTAGTTGCCAGCATTCAGTTGGGCACTCTAGAGAGACTGCCGTCGACAAGACGGAGGAAGGCGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGGGATGCTACCTCGCGAGAGGACGCCAATCTCTTAAAACCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTTGCAACACCCGAAGTCGGTGAGGTAACCGCAAGG，
Sequencing result is:
1367bp;The accession number of GeneBank is HQ585418.
2. prevent and treat the preparation method of the short genus bacillus biological pesticide of fungal diseases of plants, it is characterized in that comprising following operation steps:
(1) short genus bacillus is inoculated in the 250ml triangular flask of every bottled 50ml beef extract-peptone (NA) nutrient solution, 29 ℃ of shaking culture 24h, shaking speed 170.00r/min makes seed liquor;
Beef extract-peptone (NA) culture medium prescription is peptone 1.0%, sodium-chlor 0.5%, extractum carnis 0.5%, and initial pH value is 7.2-7.4,121 ℃ of sterilization 20min;
(2) in 5 liters of automatic fermentor tanks, fermention medium tinning 75%, by 10% inoculum size inoculation seed liquor, ventilation 3NL/min cultivates 36h down in 29 ℃, puts jar, and fermented liquid is behind the microscopy counting, and regulating concentration is 10
8-10
10Cell/ml, aseptic subpackaged, the short fermentation of bacillus liquid that obtains is the biological pesticide that contains short genus bacillus;
Described fermentative medium formula is glucose 1.00%, peptone 2.23%, dipotassium hydrogen phosphate 0.01%, magnesium sulfate heptahydrate 0.05%, and initial pH value is 7.50, real jar of sterilization.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104762326A (en) * | 2015-02-09 | 2015-07-08 | 海南大学 | Chemical transferring method for transferring hpalXoo gene into brevibacillus brevis HAB-5 |
| CN104894006A (en) * | 2015-05-12 | 2015-09-09 | 贵阳医学院 | New brevibacillus brevis strain, cultivation method and application of new brevibacillus brevis strain |
| CN105838643A (en) * | 2016-05-03 | 2016-08-10 | 唐山市畜牧水产品质量监测中心 | Bacillus subtilis JTFM1001 and application thereof to prevention and control of corn aflatoxin contamination |
| CN112980748A (en) * | 2021-05-07 | 2021-06-18 | 河北萌帮生物科技有限公司 | Brevibacillus brevis, application and method for producing humic acid by converting lignite |
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| CN104894006A (en) * | 2015-05-12 | 2015-09-09 | 贵阳医学院 | New brevibacillus brevis strain, cultivation method and application of new brevibacillus brevis strain |
| CN104894006B (en) * | 2015-05-12 | 2017-09-15 | 贵阳医学院 | New Brevibacillus brevis bacterial strain, cultural method and its application |
| CN105838643A (en) * | 2016-05-03 | 2016-08-10 | 唐山市畜牧水产品质量监测中心 | Bacillus subtilis JTFM1001 and application thereof to prevention and control of corn aflatoxin contamination |
| CN105838643B (en) * | 2016-05-03 | 2019-11-05 | 唐山市畜牧水产品质量监测中心 | Bacillus subtilis JTFM1001 and its application in prevention and control corn aflatoxin contamination |
| CN112980748A (en) * | 2021-05-07 | 2021-06-18 | 河北萌帮生物科技有限公司 | Brevibacillus brevis, application and method for producing humic acid by converting lignite |
| CN112980748B (en) * | 2021-05-07 | 2022-04-12 | 河北萌帮生物科技有限公司 | Brevibacillus brevis, application and method for producing humic acid by converting lignite |
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