CN101633941B - Method for preparing biopesticide tetramycin and application - Google Patents
Method for preparing biopesticide tetramycin and application Download PDFInfo
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- CN101633941B CN101633941B CN2008100125035A CN200810012503A CN101633941B CN 101633941 B CN101633941 B CN 101633941B CN 2008100125035 A CN2008100125035 A CN 2008100125035A CN 200810012503 A CN200810012503 A CN 200810012503A CN 101633941 B CN101633941 B CN 101633941B
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- DVWJFTGEISXVSH-CWVFEVJCSA-N (1R,3S,5S,7Z,11R,12S,13Z,15Z,17Z,19Z,21R,23S,24R,25S)-21-[(2R,3S,4S,5S,6R)-4-amino-3,5-dihydroxy-6-methyloxan-2-yl]oxy-12-ethyl-1,3,5,25-tetrahydroxy-11-methyl-9-oxo-10,27-dioxabicyclo[21.3.1]heptacosa-7,13,15,17,19-pentaene-24-carboxylic acid Chemical compound CC[C@H]1\C=C/C=C\C=C/C=C\[C@@H](C[C@@H]2O[C@@](O)(C[C@H](O)[C@H]2C(O)=O)C[C@@H](O)C[C@@H](O)C\C=C/C(=O)O[C@@H]1C)O[C@@H]1O[C@H](C)[C@@H](O)[C@H](N)[C@@H]1O DVWJFTGEISXVSH-CWVFEVJCSA-N 0.000 title claims abstract description 80
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Abstract
The invention discloses a method for preparing biopesticide tetramycin from Streptomyces ahygroscopicus subsp.Wuzhouensis n.subsp, and application of a product prepared by the method in preventing and controlling diseases such as apple alternaria leaf spots and the like. The tetramycin prepared by the method improves the biological potency of the tetramycin (the biological potency of fermentation liquor is improved by 15.5 percent after 0.3 to 0.5 percent of normal propyl alcohol is added.), can prolong the quality guarantee period, improves the effect of preventing and controlling apple canker to over 95 percent, has good effect of preventing and controlling the diseases such as the apple alternaria leaf spots, tomato gray mold and the like, has effect of inhibiting yeast, gram-negative and gram positive bacteria, fungi (comprising fungal pathogens) and the like, has wide adaptability, and belongs to the environment-friendly biopesticide.
Description
Technical field
The present invention relates to a kind of by the not biological pesticide of streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicus subsp.Wuzhouensis n.subsp) generation---the improved preparation method of tetramycin and this product application in the control alternaria leaf spot of apple.
Background technology
China is large agricultural country, plants food crop more than 100,000,000 hectares throughout the year, and cotton is more than 4,800,000 hectares; Oil crops are more than 1,400 ten thousand hectares, and vegetable melon and fruit needs the control area above 300,000,000 hectares more than 1,800 ten thousand hectares throughout the year; For prevention and elimination of disease and pests; Guarantee the ordinary production of agricultural, for a long time, people use the chemical pesticide control disease and pest in a large number.The use of chemical pesticide is played useful effect to guaranteeing the farm crop ordinary production; But along with the use of a large amount of chemical pesticides for a long time; Ecotope is done great damage; Like enzymic activity reduction in Soil structure destruction, the soil, soil property and water body severe contamination or the like; Destroyed the whole ecological environment, poisonous pesticide residue severe overweight in nearly all food such as grain, vegetables, fruit, the gourd, fruit and vegetable of edible residual chemical pesticide severe overweight causes person poultry poisoning's incident also to happen occasionally.
To China's WTO accession, developed country adjusts the technical threshold that has improved the product import one after another, and the influence that agricultural-food receive stands in the breach.Only the agricultural byproducts loss that closed down by external " green barrier " is annual just up to 7,000,000,000 dollars in China.
Developing non-harmful biological pesticide is the main path that solves existing agricultural chemicals and export of farm produce problem.Biological pesticide belongs to environmentally friendly product; Use biological pesticide; Not only can reduce the use of chemical pesticide and chemical fertilizer, realize the high-quality and safety production of agricultural-food effectively, promote the economic value added of agricultural-food; And can be agricultural products in China outlet and create favorable conditions, greatly strengthen the international competitiveness of agricultural products in China.China is advanced green agriculture industry development and developing rural economy, increases farmers' income, promotes the rural area prosperity to have important prograding.
Alternaria leaf spot of apple main harm exhibition leaf young leaflet tablet soon also can endanger the tender tip and fruit.The scab initial stage is the brown point, is expanded into the sorrel circle spot of 5～6 millimeters of diameters later on, the edge puce, and there is a Vandyke brown point at the center, and Vandyke brown ring grain, shape such as frogeye are arranged around it.During wet weather, the scab pros and cons all can produce blackish green mustiness thing.In the morbidity later stage, scab continues to be expanded into irregular shape, and scab partly or entirely is pearl, scattered pore on it, and the scab that has breaks or bores a hole.In high temperature and rainy season, scab is expanded into the big spot of irregular shape brown rapidly, and blade is shrivelled to come off.The morbidity of autumn growth tender leaf is the heaviest, and dozens of even up to a hundred the scabs that differ in size often can take place on the blade, and many scabs join together together, are the cloud shape, and blade tip is withered, and the leaf curling deformity is caducous.Petiole is injured oval dun depression scab and frangible is taken place.Branch annual shoot or branch of excessive growth susceptible disease, sick branch hole skin projection, bud week blackening, depression is downright bad, crack at edge.Young fruit is killed, is that the center produces brown circle spot with the fruit, and the surface is depression slightly, and the pulp depths is not goed deep near the suberification of pulp browning, if can be made fruit rot by secondary parasitism.Alternaria leaf spot of apple be the end of the seventies to the beginning of the eighties at a kind of new apple leaf spot of China's popular, take place in a lot of areas at present, and growing trend arranged.
At present, 77 of biological pesticide (preventing and treating phytopathy, worm, grass, the plague of rats) effective constituents that China registers are occupied and are imitated 13.4% of composition.691 of products account for 7.1% of whole Pesticidal products, and preparation output adopts accounting for about 10% of biological pesticide prevention and elimination of disease and pests near 100,000 tons.Some biological pesticide preparations also become the important kind of outlet, like Avrmectin, jingganmycin and Plant hormones regulators,gibberellins.Being used for the alternaria leaf spot of apple biological pesticide is main with polyoxin, but mostly polyoxin is the import preparation, has increased cost, is badly in need of researching and developing out novel biological pesticide, solves problems such as existing biological pesticide cost is high, control rate is low.
Tetramycin is as known compound; Be by streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicussubsp.Wuzhouensis n.subsp) (" mikrobe journal " 1988 not; 28 volume (1) 85-87 pages or leaves) produce, because the biological value of tetramycin is low, the quality guaranteed period is shorter; Control effect to canker of apple fruit is not ideal enough, and is limited aspect use and drug effect; Except that the invention unit is studied the new purposes of tetramycin, do not see other unit also has tetramycin other new purposes as biological pesticide relevant report as yet.
Summary of the invention
The object of the invention is to provide the method for utilizing streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicus subsp.Wuzhouensis n.subsp) not to prepare tetramycin, further improves the control effect to canker of apple fruit; Another object of the present invention is the application of tetramycin in the control alternaria leaf spot of apple of this method preparation.
The method for preparing tetramycin with streptomyces hygroscopicus Wuzhou new subspecies (Streptomyces ahygroscopicus subsp.Wuzhouensisn.subsp) not:
The inventive method is with streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicus subsp.Wuzhouensis n.subsp) (" mikrobe journal " 1988 not; 28 volume (1) 85-87 pages or leaves) (this bacterial classification is kept at microbial strains preservation center, Liaoning Province as starting strain; The public can obtain this bacterial classification from this center in 20 years applyings date); Produce biopesticide tetramycin through the bacterial classification liquid submerged fermentation, it is characterized in that it comprises following method:
(1) preparation of seed fermentation substratum:
Contain in per 100 milliliters: soybean cake powder 0.5-1.5g, starch 0.5-1.5g, glucose 0.5-1.5g; Peptone 0.1-0.5g, sal epsom 0.01-0.05g, ammonium sulfate 0.1-0.5g; Potassium hydrogenphosphate 0.01-0.05g, lime carbonate 0.1-1.0g, sodium-chlor 0.05-0.15g; Vegetables oil 0.2-0.5g, the PH nature;
(2) seed fermentation condition and method:
Measure not streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicus subsp.Wuzhouensis n.subsp) bacterial strain by the inoculation of 5-20%; Cultivated 20-24 hour in the seed fermentation substratum in 25-35 ℃; Ventilation: 1: 0.5-1 (V/V); Rotating speed: 150-300 rev/min, it is subsequent use to produce seed fermentation liquid;
(3) preparation of submerged fermentation culture medium:
Contain in per 100 milliliters: glucose 1.0-3.0g, starch 1.0-2.0g, Semen Maydis powder 0.5-1.5g, soybean cake powder 1.0-2.0g; Peptone (or steeping water) 0.20.6g, ammonium sulfate 0.1-0.5g, sal epsom 0.01-0.05g; Potassium hydrogenphosphate 0.01-0.05g, lime carbonate 0.1-1.0g, sodium-chlor 0.05-0.15g; Vegetables oil 0.2-0.5g, the sterilization back adds the n-propyl alcohol of culture volume 0.3-0.5%, the PH nature;
(4) tetramycin preparation of fermentation liquid:
Measure above-mentioned seed fermentation liquid by the inoculation of 5-20% and carry out submerged fermentation cultivation 64-72 hour in 25-35 ℃; Ventilation: 0-12 hour early stage, 12-40 hour mid-term, later stage 40-42 hour: 1: 0.5-1 (V/V); Rotating speed: 150-300 rev/min, obtain the tetramycin fermented liquid.
The invention still further relates to tetramycin as application at the biological pesticide of preventing and treating alternaria leaf spot of apple.
Tetramycin is a kind of complex biological microbiotic, contains tetramycin A1, A2 and albonoursin and flagecidin.According to molecular weight and some other physicochemical data that nucleus magnetic resonance, infrared absorption spectrum, uv absorption spectrum and the mass spectrum of tetramycin A1 are confirmed, the molecular formula of deriving tetramycin A1 should be C
35H
52NO
13, its chemical structural formula is:
According to similar
tetramycin A1 isolation and identification method; Through molecular weight and the some other physicochemical data that nucleus magnetic resonance, infrared absorption spectrum, uv absorption spectrum and mass spectrum are confirmed, derive tetramycin B chemical structural formula and be:
According to similar tetramycin A1 isolation and identification method,, derive tetramycin C chemical structural formula and be through molecular weight and the some other physicochemical data that nucleus magnetic resonance, infrared absorption spectrum, uv absorption spectrum and mass spectrum are confirmed:
Following table
is the tetramycin antimicrobial spectrum
| Indicator | The bacterial classification source | Connect resistance | Indicator | The bacterial classification source | Connect resistance |
| Bacillus sphaericus | In little institute | --- | Intestinal bacteria | Traditional Chinese Medicine Department institute | --- |
| Pseudomonas fluorescens | In little institute | --- | Intestinal bacteria | In little institute | + |
| Gold-coloured staphylococci | In little institute | --- | Gold-coloured staphylococci | Traditional Chinese Medicine Department institute | + |
| Bacillus cereus | In little institute | --- | Bacillus subtilus | In little institute | + |
| Bacteria erwinia | In little institute | --- | Brevibacterium flavum | In little institute | + |
| Pseudomonas aeruginosa | Traditional Chinese Medicine Department institute | --- | Mycobacterium tuberculosis | In little institute | + |
| Sarcina lutea | Traditional Chinese Medicine Department institute | ++ | The pan mould | In little institute | + |
| Yeast saccharomyces cerevisiae | In little institute | ++ | Fusarium graminearum | In little institute | + |
| Unusual debaryomyces hansenii | In little institute | --- | The beautiful broomcorn millet Helminthosporium sp. that belongs to | In little institute | - |
| Fission yeast | In little institute | ++ | Chain lattice spore is mould | In little institute | - |
| Rhodothece rubra | In little institute | ++ | The cotton anthracnose bacterium | In little institute | - |
| Ka Er Qi Shi uncle yeast | In little institute | ++ | The fusarium graminaria bacterium | In little institute | + |
| Rhodotorula glutinis | In little institute | ++ | Aspergillus fumigatus | In little institute | + |
| Candida albicans | In little institute | ++ | Flavus | In little institute | + |
| Candida krusei | In little institute | + | Penicillium crysogenum | In little institute | + |
| Candida lipolytica | In little institute | +++ | Wood is mould | In little institute | - |
| The K yeast | In little institute | ++ | Aspergillus oryzae | In little institute | - |
| Italy's mould | In little institute | +++ | Black mold | In little institute | + |
| The pears phyllosticta | In little institute | ++ | The post spore is mould | In little institute | - |
| Ramularia panacicola | The Chinese Academy of Agricultural Sciences | ++ | Sturdy post is at a distance from spore | In little institute | - |
The tetramycin biological detecting method:
Method: cup-plate method
Indicator: saccharomyces sake (Saccharomyces sake)
Substratum: upper strata: PDA
Lower floor: water agar
Standard substance: the pure article of tetramycin A
Product is demarcated indicator: tetramycin toxicological experiments such as alternaria leaf spot of apple bacterium, Valsa mali:
(1) tetramycin small white mouse and big white mouse acute toxicity:
(2) tetramycin is to the influence of CHL-1 cell SCE frequency
Table 1: tetramycin is to the influence (disactivation system) of CHL cell SCE frequency
Table 2: tetramycin is to the influence (activation system) of CHL cell SCE frequency
The experimental result that influences of tetramycin CHL cell SCE frequency shows: 5 concentration of tetramycin all do not cause the increase of SCE frequency.Through statistical procedures and negative control than equal P＞0.1.The Bap metabolism activation has significantly brought out the SCE frequency to be increased, with negative control than P＜0.001, the difference highly significant.Above result shows, 5 concentration of tetramycin do not see all and cause that the SCE frequency increases that the result can be considered the mutagenesis feminine gender.
(3) micronucleus test of tetramycin
Tetramycin is to the influence of micronuclei in mice
Experimental result shows: tetramycin sample and negative control be P＞0.1 relatively all, do not have statistical significance.Positive control and negative control are than P＜0.001, and there were significant differences.Above result shows that tetramycin does not cause the increase of micronucleus, and the result is negative.
(4) tetramycin dermal toxicity and lagophthalmos irritation test
Tetramycin is applied in the big white mouse belly of removing belly wool, observed 7 days, the result proves skin LD50 more than 4000mg/kg, shows that the tetramycin dermal toxicity is lower, and the local skin of smearing tetramycin is not seen infringement;
Tetramycin is mixed with the 10mg/ml clear liquid, drips one of 5 large ear rabbit left eye, right eye drips saline water, drips twice every day, and for three days on end, the drug withdrawal continued was observed 3 days, and the result shows that tetramycin is to the lagophthalmos nonirritant.
(5) tetramycin rat cumulative toxicity
1. tetramycin is mixed with 400mg/ml concentration, uses distilled water diluting 40mg/ml, 20mg/ml, 10mg/ml again, PH is 5.5.
2. be that 4000mg/kg is a benchmark with tetramycin to rat LD50, establish three dosages, 1/5LD50 (800mg/kg), 1/10LD50 (400mg/kg), 1/20LD50 (200mg/kg).
3. rat is 80, and male and female half and half evenly are divided into four groups, and each 10 of every group of male and female compare with zero(ppm) water, and every day, gastric infusion was 1 time, continuous 28 days.
4. put to death half animal after the drug withdrawal, male and female half and half, second half animal is observed the back execution of two weeks.
The result shows: each dose groups animal does not all have death.Thereby cumulative coefficient calculation result is 5.6. find out tetramycin be a little less than the property accumulated material.
(6) tetramycin Salmonella reversion test
Carry out the test of tetramycin according to the Ames flat board method of mixing, the result is following:
The result shows from table: do not adding the S9 activation and adding under the S9 activatory condition, five dosage of tetramycin do not have influence to time parameter of four strain Salmonella typhimurium TA97, TA98, TA100, TA102, and return parameter naturally and do not have significant difference;
Do not adding the S9 activation and adding under the S9 activatory condition, when the tetramycin high dosage reached the 5mg/ ware, the parameter that returns of four strain Salmonella typhimuriums is increase, this presentation of results: the mutagenesis result who observes tetramycin with Salmonella reversion test was negative.
Tetramycin is as the application test example of biological pesticide at the control alternaria leaf spot of apple
1, experiment and demonstration
Test place: Liquan County, Xianyang, Shanxi province city
Trial crops and kind: apple, kind: Fuji apple
Controlling object: alternaria leaf spot of apple (Marssonina mali (P.Henn.))
Test objective: in order to verify tetramycin control alternaria leaf spot of apple control effect, effective dose and to the security of apple etc.
Envrionment conditions: test arrangement is in Liquan County, Xianyang, Shanxi province city, 10 mu of orchard areas, 12 years green apple trees, seeding row spacing 2 * 3m
2, soil type is native Lou soil, and organic content is 1.05%, and pH value is neutral.
Test medicine: tetramycin
Positive control medicament: 10% polyoxin B wettable powder
Consumption and processing numbering:
| Handle numbering | Medicament | Dispenser dosage | Formulation rate (effective constituent amount) |
| (1) | Tetramycin | 800 times | 1.88mg/kg |
| (2) | Tetramycin | 1000 times | 1.5mg/kg |
| (3) | Tetramycin | 1200 times | 1.25mg/kg |
| (4) | Positive control 10% polyoxin B | 1000 times | 100mg/kg |
| (5) | The clear water contrast | - | - |
Method of use: adopt plants stems foliar spray mist to handle
Test period and spraying times: in May, 2006-August, totally 9 dispensers
Investigation method: stop to take out living phase and autumn growth in the spring tip and stop to take out the living phase and respectively carry out a preventive effect investigation.
2 strains are investigated in every sub-district, every differentiation East, West, South, North, in five directions investigation young sprouts take out whole blades of giving birth to 5 vigorous current-year branches, write down total number of sheets, the sick number of sheets, scab number.
Stage division: 0 grade: no scab;
1 grade: lesion area accounts for below 10% of whole leaf area;
3 grades: lesion area accounts for the 11-25% of whole leaf area;
5 grades: lesion area accounts for the 26-40% of whole leaf area;
7 grades: lesion area accounts for the 41-65% of whole leaf area;
9 grades: lesion area accounts for more than 66% of whole leaf area.
The drug effect method of calculation:
In the formula: CK0, CK1 are respectively the forward and backward disease index of blank district dispenser; PT0, PT1 are respectively the forward and backward disease index of chemicals treatment district dispenser.
Result and conclusion:
1, result
(1) the spring tip stops to take out living phase tetramycin control alternaria leaf spot of apple test-results
(2) autumn growth stops to take out living phase tetramycin control alternaria leaf spot of apple test-results
2, conclusion
(1) duration of test is learnt through systematic observation, supplies reagent agent tetramycin in the test dose scope, apple tree to be grown and has no adverse effects, and fruit tree growth is vigorous;
(2) through test; We think that tetramycin has remarkable prevention effect to alternaria leaf spot of apple; Its 800-1000 doubly dilutes; Be starkly lower than at effective ingredient consumption (1.25-1.88mg/kg) under the situation of positive control 10% polyoxin B (100mg/kg), the control effect that stops to take out the living phase at the spring tip reaches the control effect of positive control; The control effect that autumn growth stops to take out the living phase also reaches the control effect of positive control, and wherein 800 times of diluent preventive effects of tetramycin are higher than positive control, and effective ingredient consumption is starkly lower than positive control.
Suggestion is carried out large-scale demonstration to it and is promoted.Recommended dose is a 800-1000 times of diluent, begins to spray the medicine control in alternaria leaf spot of apple early period of origination (the first tenday period of a month in May), and about 10-14 days spray medicines once spray continuously and are advisable for 3-4 time at interval.
Through above test-results, prove the present invention and present compared with techniques, have following beneficial effect:
1, known tetramycin generation bacterium is the strain actinomycetes that Liaoning Academy of Microorganism Sciences screens from soil; Called after is streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicus subsp.Wuzhouensis n.subsp) " mikrobe journal " 1988 not; 28 volume (1) 85-87 pages or leaves; This bacterial classification is kept at microbial strains preservation center, Liaoning Province, and the public can obtain this bacterial classification from this center in 20 years applyings date.
2, tetramycin is significantly less than under the situation of positive control 10% polyoxin at formulation rate; 800 times of dilute samples are prevented and treated canker of apple fruit when the spring tip stops to take out living phase effect is higher than polyoxin; Average preventive effect reached more than 83% when the spring tip stopped to take out living phase, and control effect is higher than or is equal to the biological pesticide that present big area is promoted.Tetramycin is further promoted, not only can be improved preventive effect, and can be used alternatingly, slow down the generation of resistance with the other biological agricultural chemicals;
3, tetramycin has broad spectrum; To yeast, Gram-negative, positive bacteria and fungi (comprising pathogenic fungi) etc. are had restraining effect; Prove with field test through indoor: the canker of apple fruit control effect is brought up to more than 95%, alternaria leaf spot of apple, rice blast and graw mold of tomato are also had good control effect;
4, tetramycin is the produced simultaneously microbiotic that contains 4 kinds of compositions of a strain bacterium, belongs to environmentally friendly agricultural chemicals, and multiple microbiotic synergy, can reduce the use of other agricultural chemicals, has the certain significance to safeguarding ecotope;
5, tetramycin is a low toxicity through evidence.Acute toxicity LD50 is 4000mg/kg, all no abnormal reaction of skin and MMi, and Salmonella reversion test, SCE mutagenesis of CHL-1 cell and micronucleus test are all negative.The accumulation toxicity test check result shows the equal Non Apparent Abnormality of each organ, and cumulative coefficient is greater than 5.6, and more than explanation tetramycin toxicity is very low, and acute toxicity and salt and sodium bicarbonate are similar;
6, utilize the improved tetramycin preparation method of the present invention, not only improved the biological value (after adding the n-propyl alcohol of 0.3-0.5%, the fermented liquid biological value has improved 15.5%) of tetramycin, and the quality guaranteed period is increased.Produce and still adopt universal fermentation equipment, technology is simple, and the cycle is short.There is not three wastes problem in the raw materials for production low price in the production process, can not cause environmental pollution;
Embodiment
Through specific embodiment given below, can further understand the present invention.But they are not the restriction to protection domain of the present invention.
Embodiment 1: the tetramycin preparation of fermentation liquid
The present invention passes through the preferred culture medium prescription of orthogonal test:
(1) seed fermentation substratum: contain in per 100 milliliters: soybean cake powder 1.0g, starch 1.0g, glucose 0,7g; Peptone 0.5g, sal epsom 0.03g, ammonium sulfate 1.0g, potassium hydrogenphosphate 0.02g; Lime carbonate 0.7g, sodium-chlor 0.1g, vegetables oil 0.2g, PH nature;
(2) submerged fermentation culture medium: contain in per 100 milliliters: glucose 2.5g, starch 1,5g, Semen Maydis powder 1.0g; Soybean cake powder 2,0g, peptone (or steeping water) 0.5g, ammonium sulfate 0.2g; Sal epsom 0.03g, potassium hydrogenphosphate 0.02g, lime carbonate 0.7g, sodium-chlor 0.1g; Vegetables oil 0.2g, the n-propyl alcohol of culture volume 0.4% is added in the sterilization back, the PH nature.
The present invention optimizes fermentation condition:
(1) preparation of seed fermentation liquid: measure not streptomyces hygroscopicus Wuzhou subspecies (Streptomycesahygroscopicus subsp.Wuzhouensis n.subsp) bacterial strain by the inoculation of 5-20%; Cultivated 20-24 hour in the seed fermentation substratum in 25-35 ℃; Ventilation: 1: 0.5-1 (V/V); Rotating speed: 150-300 rev/min, it is subsequent use to produce seed fermentation liquid;
(2) tetramycin preparation of fermentation liquid: measure above-mentioned seed fermentation liquid by the inoculation of 5-20% and carry out the submerged fermentation cultivation in 25-35 ℃; Ventilation: 0-12 hour early stage, 12-40 hour mid-term, later stage 40-42 hour: 1: 0.5-1 (V/V); Rotating speed: 150300 rev/mins; Inoculum size: 5-20% fermentation period: 64-72 hour, obtains the tetramycin fermented liquid.
Embodiment 2-5: tetramycin control alternaria leaf spot of apple application example
Claims (1)
1. the preparation method of a biopesticide tetramycin; Be to adopt not that streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicus subsp.Wuzhouensis n.subsp) through the biological pesticide---tetramycin that the bacterial classification liquid submerged fermentation is produced, is characterized in that it may further comprise the steps:
(1) seed fermentation medium preparation:
Contain in per 100 milliliters: soybean cake powder 0.5-1.5g, starch 0.5-1.5g, glucose 0.5-1.5g; Peptone 0.1-0.5g, sal epsom 0.01-0.05g, ammonium sulfate 0.1-0.5g; Potassium hydrogenphosphate 0.01-0.05g, lime carbonate 0.1-1.0g, sodium-chlor 0.05-0.15g; Vegetables oil 0.2-0.5g, the PH nature;
(2) seed fermentation condition and method:
Measure not streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicus subsp.Wuzhouensis n.subsp) bacterial strain by the inoculation of 5-20%; Cultivated 20-24 hour in the seed fermentation substratum in 25-35 ℃; Ventilation: 1: 0.5-1 (V/V); Rotating speed: 150-300 rev/min, it is subsequent use to produce seed fermentation liquid;
(3) submerged fermentation culture medium preparation:
Contain in per 100 milliliters: glucose 1.0-3.0g, starch 1.0-2.0g, Semen Maydis powder 0.5-1.5g, soybean cake powder 1.0-2.0g; Peptone or steeping water 0.2-0.6g, ammonium sulfate 0.1-0.5g, sal epsom 0.01-0.05g; Potassium hydrogenphosphate 0.01-0.05g, lime carbonate 0.1-1.0g, sodium-chlor 0.05-0.15g; Vegetables oil 0.2-0.5g, the sterilization back adds the n-propyl alcohol of culture volume 0.3-0.5%, the PH nature;
(4) tetramycin fermented liquid preparation:
Measure above-mentioned seed fermentation liquid by the inoculation of 5-20% and carry out submerged fermentation cultivation 64-72 hour in 25-35 ℃; 0-12 hour early stage, 12-40 hour mid-term, later stage 40-42 hour; Ventilation: 1: 0.5-1 (V/V), obtains the tetramycin fermented liquid by rotating speed: 150-300 rev/min.
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