CN101422170A - Streptomyces globisporus microbial agent for controlling Alternaria alternate and preparation method thereof - Google Patents
Streptomyces globisporus microbial agent for controlling Alternaria alternate and preparation method thereof Download PDFInfo
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- CN101422170A CN101422170A CNA2008102335019A CN200810233501A CN101422170A CN 101422170 A CN101422170 A CN 101422170A CN A2008102335019 A CNA2008102335019 A CN A2008102335019A CN 200810233501 A CN200810233501 A CN 200810233501A CN 101422170 A CN101422170 A CN 101422170A
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Abstract
The invention discloses a biological agent for controlling a tobacco brown spot. The production strain of the biological agent is sorted and named as Streptomyces globisporus AM6 and the preserving number thereof is CGMCCNo.1701. The prescriptions of the solid and the liquid mediums of the strain are as follows: (1) the incline medium of a test tube is as follows: 200g of fresh potatoes, 20g of dextrose, 17 to 20g of agar, 1000ml of distilled water as well as a natural pH; (2) the prescription of a liquid fermentation medium is as follows: 10.0g of bean powder, 20.0g of cane sugar, 2g of dipotassium hydrogen phosphate, 0.02g of green copperas, 0.5g of calcium carbonate, 0.05g of bitter salt, 1000ml of tap water as well as a natural pH. The invention is prepared into the biological agent by the test tube culturing of the strain and the fermentation culturing of a table concentrator. Test results show that the biological agent provided by the invention has excellent controlling effects on the tobacco brown spot.
Description
Technical field
The present invention relates to biopesticide, more particularly, the present invention relates to a kind of microbial inoculum of preventing and treating Alternaria alternate and preparation method thereof.
Background technology
Tobacco is that the important leaf of China is used economic crops, is one of the most serious crop of disease hazard, and from being seeded into results, whole process all can be subjected to infecting of pathogen.Alternaria alternate (Tobacco BrownSpot Disease) is the ripe important leaf diseases of later stage of tobacco leaf, generally takes place in each cigarette district, and be the main disease of China tobacco.In recent years, the rust that economize in the Hunan of China, Anhui, Guizhou, Yunnan, Shaanxi, Liaoning, Zhejiang, Guangdong, Fujian, Heilungkiang, Jilin etc. is development to some extent also, become the maximum a kind of leaf spot that causes harm, the general time incidence of disease is 20%～30%, the serious incidence of disease reaches 90%, reduce the output value reaches more than 50%, bigger to output, quality influence.
Alternaria alternate is a kind of typical aeroborne disease, the present domestic prophylactico-therapeutic measuress such as chemical pesticide that when control, mainly wait agricultural measures and heavy dose with the health care cultivation, and shortage can be for the resistant variety of zone cultivation; Use dimethachlon to prevent and treat for many years, cause drug cost soaring year by year, control efficiency is progressively weakening, and some disease funguses develop immunity to drugs, and difficulty of prevention and cure also strengthens thereupon.
Because chemical agent very easily forms poisoning, also be subject to rainwash during dispenser and lost efficacy, and shortage high-efficiency low-toxicity chemical control medicament, continuous use easily causes residue of pesticide, to the baneful influence that environment causes, polluted source causes person poultry poisoning, death, simultaneously pathogen is developed immunity to drugs, cause its use to be subjected to certain limitation.On the other hand, Alternaria alternate is a tobacco maturing stage disease, since more and more higher to the requirement of tobacco safety, concerning tobacco cultivation itself, just have higher requirement, particularly residual chemical agent is arranged for not quite suitable employing of the control of this maturing stage disease of Alternaria alternate.This is the urgency technical barrier to be solved in this area.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art part, the microbial inoculum of a kind of effective, nontoxic, safety, noresidue, control Alternaria alternate easy to use is provided.Simultaneously, the present invention also provides a kind of preparation method of described microbial inoculum.
Purpose of the present invention is achieved by following technical proposals.
A. the invention provides a kind of microbial inoculum of preventing and treating Alternaria alternate, the actinomycetes that this microbial inoculum adopts are classification called after styreptomyces globispotus strain (Streptomyces globisporus) AM6, and deposit number is CGMCC No.1701.
Production bacterial strain styreptomyces globispotus strain of the present invention (Streptomyces globisporus) AM6, separation is on the typical Alternaria alternate scab in Mengzi, Yunnan, indoor excised leaf sessile drop method is measured and is found that 100 times of culture fluids that carry disease germs all reach 100% to the preventive effect of rust, plate measure to find that the AM6100 culture fluid that doubly carries disease germs is 100% to the inhibitory action of strong pathogenicity germ, the inhibition effect of its sterilization filtrate is 81.8%, its antifungal mechanism mainly is to produce antagonistic substance, microscopic examination shows that AM6 can make germ spore and mycelia distortion, cell wall rupture, protoplasm is revealed.This bacterial strain has following feature:
1. on Gause I agar and the asparagine agar 28 ℃ cultivate 7～10 days after, strains A M6 substrate mycelium does not have tabula, does not rupture, no sporangiocyst; The aerial hyphae branch is more; Fibrillae of spores is straight or crooked, and idol is grown thickly; Spore sphere or oval, smooth surface.That grows on 8 kinds of medium the results are shown in Table 1.
The cultural characteristic of table 1. strains A M6 on 8 kinds of medium
2. full cell hydrolyzate contain the LL-diaminopimelic acid (LL-DAP, LL-Diaminopimelicacid), glycine, sugared atypism (sugared type C); Contain phosphatidyl-ethanolamine (PE), phosphate lipid is the II type.
3. have following physio-biochemical characteristics: propylhomoserin enzyme feminine gender, can on cellulose, grow, produce class melanin, do not produce H
2S, liquefy gelatin, peptonized milk does not solidify milk, hydrolyzed starch, reduction nitrate utilizes L-arabinose, L-rhamnose, D-glucose, D-mannose, D-fructose, D-wood sugar, galactose, does not utilize mannose, raffinose, inositol.
B. the invention provides the preparation method of styreptomyces globispotus strain (Streptomyces globisporus) AM6 biologic product, this method adopts following steps:
1. the test tube kind is cultivated
The test tube slant culture medium prescription is: fresh potato 200g, glucose 20g, agar 17-20g, distilled water 1000ml, pH nature.
Styreptomyces globispotus strain (Streptomyces globisporus) AM6 streak inoculation to the test tube slant medium, 28 ℃ ± 1 ℃ constant temperature culture 4～5 days, is obtained the test tube kind.
2. liquid fermentation and culture
The liquid fermentation medium prescription is: analysis for soybean powder 10.0g, sucrose 20.0g, dipotassium hydrogen phosphate 2g, ferrous sulfate 0.02g, calcium carbonate 0.5g, magnesium sulfate 0.05g, running water 1000ml, pH nature.
Every test tube slant is added sterile water 5-10ml, scrape conidium, shake up with oese, form spore suspension, volume ratio with 0.2% inserts dress and cultivates in the triangular flask that liquid measure is 40%～60% (volume of dress culture fluid and the volumetric ratio of container), cultivates smear 7～8 days in 150～200 rev/mins of speed oscillations of 28 ℃ ± 1 ℃ constant temperature, microscopy, produce a large amount of spores, plate count is measured spore content, when every milliliter of spore content reaches 10,000,000,000 when above, stop fermentation, room temperature left standstill 12 hours.
3. preparationization
The microbial inoculum of styreptomyces globispotus strain (Streptomyces globisporus) AM6 is the liquid fermentation preparation, the preparation method is as follows: the bacterium liquid of fermentation after leaving standstill adds that 0.5% sodium carboxymethylcellulose, 0.2% benzoic acid are received, 5% dimethachlon, when 50 rev/mins of rotating speeds quicken to be stirred to 250 rev/mins gradually, constant speed continuous stirring 15 minutes, the colloid mill fragmentation, cross 200 order fluids sieve, in the plastics spiral cover bottle of packing into.
1 year this liquid fermentation preparation shelf-life, need fully shake up when using.
Compared with prior art, the present invention has following beneficial effect:
1. the present invention utilizes the biological control technology, has succeeded in developing the microbial inoculum of a kind of efficient, nontoxic, safety, noresidue, control Alternaria alternate easy to use.This all has good result to usage amount, minimizing environmental pollution, the reduction residue of pesticide that alleviate disease hazard, minimizing chemical pesticide;
2. the present invention is to overcoming the pesticide resistance of disease fungus, improve quality of tobacco, strengthen tobacco field ecosystem stability, ensure that the sustainable development of tobacco industry has important practical significance and wide application prospect, will in the foreign trade competition in future, occupy perch.
The explanation of preservation biomaterial
Bacterial strain that the present invention adopts, be positioned at China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) preservation of Zhong Guan-cun, BeiJing, China on April 24th, 2006, classification called after styreptomyces globispotus strain (Streptomyces globisporus) AM6, deposit number is CGMCC No.1701.
Embodiment
By specific embodiment given below and Application Example, can further be well understood to the present invention.But they are not the qualifications to protection domain of the present invention.
Embodiment 1
---liquid fermentation agent preparation method 1
(1) strain preparation
Test tube slant medium (fresh potato 200g, glucose 20g, agar 17-20g, distilled water 1000ml, pH nature are arrived in the streak inoculation of styreptomyces globispotus strain (Streptomyces globisporus) AM6 bacterial strain.) on, 28 ℃ of constant temperature culture 4～5 days, obtain the test tube kind.
(2) liquid fermentation and culture
Every test tube slant is added sterile water 10ml, scrape conidium with oese, shake up, form spore suspension, volume ratio with 0.2% inserts 300ml culture fluid (analysis for soybean powder 10.0g is housed, sucrose 20.0g, dipotassium hydrogen phosphate 2g, ferrous sulfate 0.02g, calcium carbonate 0.5g, magnesium sulfate 0.05g, running water 1000ml, pH nature.) the 500ml triangular flask in, cultivated 7～8 days in 150～200 rev/mins of speed oscillations of 28 ℃ of constant temperature, smear, microscopy produces a large amount of spores, plate count is measured spore content, when every milliliter of spore content reaches 10,000,000,000 when above, stops to ferment, room temperature left standstill 12 hours.
(3) preparationization
The microbial inoculum of biocontrol bacterial strain styreptomyces globispotus strain of the present invention (Streptomyces globisporus) AM6 is the liquid fermentation preparation, and the preparation method is as follows:
The bacterium liquid of fermentation after leaving standstill adds that 0.5% sodium carboxymethylcellulose, 0.2% benzoic acid are received, 5% dimethachlon, when 50 rev/mins of rotating speeds quicken to be stirred to 250 rev/mins gradually, constant speed continuous stirring 15 minutes, colloid mill fragmentation, cross 200 order fluids sieve, in the 250ml plastics spiral cover bottle of packing into.Promptly get liquid bacterial agent of the present invention.
Embodiment 2
---liquid fermentation formulation preparation method 2
(1) strain preparation
With embodiment 1.
(2) liquid fermentation and culture
Being divided into shake flask fermentation cultivation and ferment tank cultivates.The shake flask fermentation cultivation foreshortens to 4～5 beyond the highest heavens except that the cultivation and fermentation time, liquid fermentation and culture in all the other the same examples observes that mycelia is richer, newborn a large amount of spores on the mycelia, and plate count is measured spore content, when every milliliter of spore content reaches more than 6,000,000,000, obtain the female kind of fermentation tank.Plant by 0.2% volume ratio access as mother again culture fluid (analysis for soybean powder 10.0g, sucrose 20.0g, dipotassium hydrogen phosphate 2g, ferrous sulfate 0.02g, calcium carbonate 0.5g, magnesium sulfate 0.05g, running water 1000ml, pH nature are housed.) fermentation tank in, cultivated 6～7 days in 28 ℃ of constant temperature 150-200 rev/mins of rotating speeds, plate count is measured spore content, when every milliliter of spore content reaches 10,000,000,000 when above, stops to ferment, room temperature left standstill 12 hours.
(3) preparationization
With embodiment 1.
Application Example 1
---the same field comparative trial of microbial inoculum
1.1 materials and methods
Experimental field piece and management: choose that physical features is more smooth, fertility is consistent, field, rust district occurred frequently in former years piece.The kind of cultivation is local main breed, seeding row spacing 1.0-1.1 * 0.55-0.60 rice.Remove after the cigarette transplantation of seedlings survives, do not use beyond the fungicide of blade face no specific (special) requirements.Other carries out cultivation management by local conventional measure.
Handle and be provided with: microbial inoculum of the present invention (200 times), polyoxin (600 times), dimethachlon (Si Peisi board, 500 times of 40% wetting powders), Chi Bante (500 times), 1 clear water blank, totally 5 processing, 0.5 mu of every processing area.
Application method: each medicament begins to spray medicine at Alternaria alternate their early stage when produce finding that light symptoms occurs (or land for growing field crops), with workers and peasants' 16 type knapsack sprayers, convert water by the experimental scheme concentration requirement and mix, in cigarette strain leaf positive and negative even spraying, every spray in 7～10 days 1 time, execute altogether 2 times.
Observe and record: each rust state of an illness of handling of investigation respectively, the 1st preceding 1 day of medication of spraying or investigated the investigation in the 7th day after the last medication 1 time the same day.Investigation is carried out by (YC/T39-1996) tobacco business standard.When sampling investigation, by 5 samplings, promptly 5 of each sub-districts decide strain 10 strains of taking a sample and are done the fixed point investigation, are unit with the blade, respectively the incidence of classification investigation record appointment leaf position blade.Calculate disease index and preventive effect.
1.2 result and analysis
The same field comparative trial of different medicaments shows that 4 kinds of medicaments of confession examination have bigger difference to the control efficiency of Alternaria alternate.After the dispenser 2 times, better with dimethachlon and microbial inoculum of the present invention to the control efficiency of rust, be respectively 74.6% and 73.6%, relatively poor with the preventive effect of polyoxin, be 66.2%.From for 2 kinds of biologic products of examination to comparison shows that of rust preventive effect, microbial inoculum of the present invention has preferably that development prospect sees Table 2.
Table 2. microbial inoculum same field comparative test result
Application Example 2
---the experiment and demonstration of microbial inoculum
Adopt the multiple spot small-scale way of promotion to carry out, in Hongta District, the tobacco planting district production popularizing department on 4 ground such as Mengzi County, Binchuan County, Milei County is for relying on, 200 mu the experiment and demonstration that adopted polyoxin, microbial inoculum unified plan of the present invention, and compare with dimethachlon, to make blank with the about 0.5 mu field piece of sheet area.The result shows, takes place under the not serious prerequisite in disease, and polyoxin, streptomycete preparation and contrast medicament dimethachlon difference are not remarkable, see Table 3.Conscientiously carrying out under the condition of observing and predicting, when the state of an illness is not serious, but microbial inoculum large tracts of land of the present invention is promoted the use of.
Table 3 control Alternaria alternate experiment and demonstration effect
Claims (3)
1. microbial inoculum of preventing and treating Alternaria alternate, it is characterized in that: the production bacterial strain of this microbial inoculum is styreptomyces globispotus strain (Streptomyces globisporus) AM6, and preserving number is: CGMCCNo.1701.
2. microbial inoculum according to claim 1, the liquid of described bacterial strain and solid culture based formulas are as follows: 1. the test tube slant culture medium prescription is: fresh potato 200g, glucose 20g, agar 17-20g, distilled water 1000ml, pH nature; 2. the liquid fermentation medium prescription is: analysis for soybean powder 10.0g, sucrose 20.0g, dipotassium hydrogen phosphate 2g, ferrous sulfate 0.02g, calcium carbonate 0.5g, magnesium sulfate 0.05g, running water 1000ml, pH nature.
3. preparation method who prevents and treats the microbial inoculum of Alternaria alternate is characterized in that adopting following steps:
(1) the test tube kind is cultivated
The test tube slant culture medium prescription is: fresh potato 200g, glucose 20g, agar 17-20g, distilled water 1000ml, pH nature; Styreptomyces globispotus strain (Streptomyces globisporus) AM6 streak inoculation to the test tube slant medium, 28 ℃ ± 1 ℃ constant temperature culture 4～5 days, is obtained the test tube kind;
(2) liquid fermentation and culture
The liquid fermentation medium prescription is: analysis for soybean powder 10.0g, sucrose 20.0g, dipotassium hydrogen phosphate 2g, ferrous sulfate 0.02g, calcium carbonate 0.5g, magnesium sulfate 0.05g, running water 1000ml, pH nature; Every test tube slant is added sterile water 5-10ml, scrape conidium, shake up with oese, form spore suspension, volume ratio with 0.2% inserts dress and cultivates in the triangular flask that liquid measure is 40%～60% (volume of dress culture fluid and the volumetric ratio of container), cultivates smear 7～8 days in 150～200 rev/mins of speed oscillations of 28 ℃ ± 1 ℃ constant temperature, microscopy, produce a large amount of spores, plate count is measured spore content, when every milliliter of spore content reaches 10,000,000,000 when above, stop fermentation, room temperature left standstill 12 hours;
(3) preparationization
The microbial inoculum of styreptomyces globispotus strain (Streptomyces globisporus) AM6 is the liquid fermentation preparation, the preparation method is as follows: the bacterium liquid of fermentation after leaving standstill adds that 0.5% sodium carboxymethylcellulose, 0.2% benzoic acid are received, 5% dimethachlon, when 50 rev/mins of rotating speeds quicken to be stirred to 250 rev/mins gradually, constant speed continuous stirring 15 minutes, the colloid mill fragmentation, cross 200 order fluids sieve, in the plastics spiral cover bottle of packing into; Promptly make the microbial inoculum of required control Alternaria alternate.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103695350A (en) * | 2013-12-18 | 2014-04-02 | 肇庆市真格生物科技有限公司 | Streptomyces griseus CCCQ171 and application thereof |
| CN105475360A (en) * | 2015-12-28 | 2016-04-13 | 国家海洋局第三海洋研究所 | Composition containing bacillus subtilis and dimethachlon and application thereof |
| CN107354103A (en) * | 2017-04-21 | 2017-11-17 | 浙江师范大学 | Streptomycete Streptomyces lunalinharesii ZJNU968 bacterial strains and its application |
| CN117121923A (en) * | 2023-10-25 | 2023-11-28 | 云南五佳生物科技有限公司 | Composition for preventing and treating tobacco brown spot, preparation method and application |
Family Cites Families (2)
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| CN1212773C (en) * | 2002-11-22 | 2005-08-03 | 安徽林苑虫草研究所 | Process for producing Beauveria globisporus non-woven fibric ribbon |
| CN1233818C (en) * | 2003-05-22 | 2005-12-28 | 郑爱萍 | Strain of streptomycete for preventing sheath blight of rice |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695350A (en) * | 2013-12-18 | 2014-04-02 | 肇庆市真格生物科技有限公司 | Streptomyces griseus CCCQ171 and application thereof |
| CN105475360A (en) * | 2015-12-28 | 2016-04-13 | 国家海洋局第三海洋研究所 | Composition containing bacillus subtilis and dimethachlon and application thereof |
| CN105475360B (en) * | 2015-12-28 | 2019-01-11 | 国家海洋局第三海洋研究所 | Composition and application containing bacillus subtilis and dimethachlon |
| CN107354103A (en) * | 2017-04-21 | 2017-11-17 | 浙江师范大学 | Streptomycete Streptomyces lunalinharesii ZJNU968 bacterial strains and its application |
| CN107354103B (en) * | 2017-04-21 | 2020-05-26 | 浙江师范大学 | Streptomyces lunalinharesii ZJNU968 strain and application thereof |
| CN117121923A (en) * | 2023-10-25 | 2023-11-28 | 云南五佳生物科技有限公司 | Composition for preventing and treating tobacco brown spot, preparation method and application |
| CN117121923B (en) * | 2023-10-25 | 2023-12-26 | 云南五佳生物科技有限公司 | Composition for preventing and treating tobacco brown spot, preparation method and application |
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