CN101633941A - Method for preparing biopesticide tetramycin and application - Google Patents

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

The preparation method of biopesticide tetramycin and application

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, the plantation food crop are more than 100,000,000 hectares throughout the year, 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, as 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.

At 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 are subjected to 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 canescence, 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 frangibility 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, as Avrmectin, jingganmycin and Plant hormones regulators,gibberellins.Be used for the alternaria leaf spot of apple biological pesticide based on polyoxin, but polyoxin mostly is the import preparation, has increased cost, is badly in need of researching and developing out novel biological pesticide, solve problems such as existing biological pesticide cost height, control rate are low.

Tetramycin is as known compound, be by streptomyces hygroscopicus Wuzhou subspecies (Streptomyces ahygroscopicussubsp.Wuzhouensis n.subsp) (" microorganism journal " 1988 not, 28 volume (1) 85-87 pages or leaves) produce, because the biological value of tetramycin is low, quality guaranteed period is shorter, prevention effect to canker of apple fruit is not ideal enough, and is limited aspect use and drug effect; Except that 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 prevention 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) (" microorganism 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 by 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, dipotassium hydrogen phosphate 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 standby 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 corn steep liquor) 0.20.6g, ammonium sulfate 0.1-0.5g, sal epsom 0.01-0.05g, dipotassium hydrogen phosphate 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, ultra-violet absorption spectrum and the mass spectrum of tetramycin A1 are determined, 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, by molecular weight and the some other physicochemical data that nucleus magnetic resonance, infrared absorption spectrum, ultra-violet absorption spectrum and mass spectrum are determined, 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 by molecular weight and the some other physicochemical data that nucleus magnetic resonance, infrared absorption spectrum, ultra-violet absorption spectrum and mass spectrum are determined:

Following table

Be 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	Department of Chinese medicine institute	??---
Pseudomonas fluorescens	In little institute	??---	Intestinal bacteria	In little institute	??+
						Gold-coloured staphylococci	In little institute	??---	Gold-coloured staphylococci	Department of Chinese medicine institute	??+
Bacillus cereus	In little institute	??---	Bacillus subtilus	In little institute	??+
						Bacteria erwinia	In little institute	??---	Brevibacterium flavum	In little institute	??+
Pseudomonas aeruginosa	Department of Chinese medicine institute	??---	Mycobacterium tuberculosis	In little institute	??+
						Sarcina lutea	Department of Chinese medicine 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	??+++	Aspergillus niger	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 every 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 product 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.Learn to handle by statistics with 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 that 5 concentration of tetramycin there is no and cause that the SCE frequency increases, and 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 physiological saline, drips twice every day, for three days on end, continues after the drug withdrawal to observe 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 distilled 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 dosage treated 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 as follows:

The result shows from table: do not adding S9 activation and adding under the S9 activatory condition, five dosage of tetramycin do not have influence to the parameter that returns 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 demonstration example of biological pesticide at the control alternaria leaf spot of apple

1, experiment and demonstration

Test site: 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 prevention 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	??-	??-

Using method: 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, and the reagent agent tetramycin grows to apple tree in the test dose scope and has no adverse effects, and fruit tree growth is vigorous;

(2) by 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 prevention effect that stops to take out the living phase at the spring tip reaches the prevention effect of positive control; The prevention effect that autumn growth stops to take out the living phase also reaches the prevention 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.

By above test-results, prove that the present invention compares with present technology, has 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) " microorganism 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 prevention 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 by indoor and field test: the canker of apple fruit prevention effect is brought up to more than 95%, alternaria leaf spot of apple, rice blast and graw mold of tomato are also had good prevention 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 mucous membrane irritation, 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

By specific embodiment given below, can further understand the present invention.But they are not limiting the scope of the 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, dipotassium hydrogen phosphate 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 corn steep liquor) 0.5g, ammonium sulfate 0.2g, sal epsom 0.03g, dipotassium hydrogen phosphate 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 standby 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, obtain the tetramycin fermented liquid.

Embodiment 2-5: tetramycin control alternaria leaf spot of apple application example

Claims (2)

1, a kind of preparation method of biopesticide tetramycin, be to adopt not that streptomyces hygroscopicus Wuzhou subspecies (Streptomycesahygroscopicus subsp.Wuzhouensis n.subsp) is characterized in that by the biological pesticide---tetramycin that the bacterial classification liquid submerged fermentation is produced it comprises following method:

(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, dipotassium hydrogen phosphate 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 standby 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 corn steep liquor 0.2-0.6g, ammonium sulfate 0.1-0.5g, sal epsom 0.01-0.05g, dipotassium hydrogen phosphate 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 ℃, 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.

2, according to the tetramycin of claim 1 preparation as the application of biological pesticide in the control alternaria leaf spot of apple.