CN104686582A - Preparation method of microorganism bacterium agent for resisting potato scab - Google Patents
Preparation method of microorganism bacterium agent for resisting potato scab Download PDFInfo
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- CN104686582A CN104686582A CN201310665843.9A CN201310665843A CN104686582A CN 104686582 A CN104686582 A CN 104686582A CN 201310665843 A CN201310665843 A CN 201310665843A CN 104686582 A CN104686582 A CN 104686582A
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Abstract
The invention discloses a preparation method of a microorganism bacterium agent. Bacillus subtilis CGMCC1.3382, Dactylosporangium sp. ACCC No.40661 and Streptomyces roseoflavus ACCC No.40400 are used as preparation strains; all the strains are prepared into fermented solutions respectively; and the fermentation solutions are mixed and then are deeply fermented to obtain the microorganism bacterium agent. The series of the strains reasonably utilize the mutual effect of microorganisms, and the product has the characteristics of strong adaptability, good stability, uneasiness in ageing, strong pathogenic bacteria antagonism and the like; and the pathogenic bacteria of the potato scab can be efficiently resisted, the growth of plants is stimulated and the quality of crops is improved, so that the aims of yield increasing and high yield are realized.
Description
Technical field
The invention belongs to agricultural and biological technical field, relate to a kind of microbial bacterial agent of antagonism deep scab of potato, specifically, relate between different strain and carry out by synergy a kind of a kind of microbial bacterial agent effectively can treating deep scab of potato that effective compound produces.
Background technology
Potato is one of important crops of China, and status is only second to paddy rice, wheat and maize, is the fourth-largest crops of China.The planting area of potato is extensive, and disease is also comparatively complicated, is wherein one of important disease of nowadays influence potato production by the microbial deep scab of potato of various plants cause of disease strepto-.
Deep scab of potato is mainly microbial by strepto-, and its mycelia is elongated, and successive segmentation generates a large amount of spore.Spore cylindrical shape, pathogenic bacteria generally invade from potato epidermis hole skin or wound via soil pore, but invade after the suberification of potato tubers epidermis more difficult.Deep scab of potato suitable morbidity under the dry situation of soil high-temperature, suitable onset temperature is 20-30 DEG C, and suitable acid-base value is pH5-8.6.Deep scab of potato affects outward appearance and the quality of potato, makes the Quality Down of potato block, reduces the commodity value of potato, makes it the market competitiveness and reduces, thus bring economic loss.
Work out the micropopulation of inhibition of potato shot hole to be an important topic of benefiting the nation and the people.
Summary of the invention
Problem to be solved by this invention be develop a kind of can the microbial bacterial agent of efficient antagonism deep scab of potato, this system fungus feature mainly strong adaptability, good stability, not easily aging, to features such as pathogen Antagonism are strong, and the interaction between Appropriate application microorganism, the production fermentation technique being equipped with high-tech produces the product of high-quality.The pathogen of the efficient antagonism deep scab of potato of this microbial bacterial agent energy, stimulating plant grows, improves the effects such as crop quality, thus reaches the object of volume increase, high yield.
The present invention measures the bacteriostasis of bacterial classification by double-layer agar technique, then measures the bacteriostasis of zymotic fluid by paper disk method, finally by the composition of response phase method determination final products.Its technical scheme is as follows: a kind of microbial bacterial agent of antagonism deep scab of potato, and it adopts bacillus subtilis (Bacillus subtilis) CGMCC1.3382, actinomycetes (Dactylosporangium sp.) ACCC No.40661, rose yellow streptomycete (Streptomyces roseoflavus) ACCC No.40400 as preparing bacterial classification; The mass percent of described bacterial classification is as follows: bacillus subtilis 30 ~ 35%, actinomycetes 30 ~ 35%, rose yellow streptomycete 33 ~ 38%.
The microbial bacterial agent of antagonism deep scab of potato of the present invention, as mix bacterium agent after it adopts bacillus subtilis (Bacillus subtilis) CGMCC1.3382 zymotic fluid, actinomycetes (Dactylosporangium sp.) ACCC No.40661 zymotic fluid, rose yellow streptomycete (Streptomyces roseoflavus) ACCC No.40400 zymotic fluid to mix, obtain through fermentation again.Wherein, described bacillus subtilis (Bacillus subtilis) CGMCC1.3382 zymotic fluid, actinomycetes (Dactylosporangium sp.) ACCC No.40661 zymotic fluid, rose yellow streptomycete (Streptomycesroseoflavus) ACCC No.40400 zymotic fluid, respective bacterial classification quantity is all not less than 10
8individual/mL.The mass percent of three's mixing is respectively 30 ~ 35%, 30 ~ 35%, 33 ~ 38%.
For technical scheme mentioned above, in preferred situation: the inoculum concentration of described mix bacterium agent through again fermenting is that mix bacterium agent accounts for 1 ~ 5% of total fermentation medium quality.
For technical scheme mentioned above, in preferred situation: the described fermentation medium again fermented is: glucose 15 ~ 30g/L, starch 10 ~ 15g/L, peptone 15 ~ 23g/L, NaCl5 ~ 10g/L, KH
2pO
40.1 ~ 0.5g/L, all the other are distilled water; PH6.0 ~ 8.0.
For technical scheme mentioned above, in preferred situation: the end-product total bacteria count that described fermentation again obtains is at least 10 × 10
8individual/mL.
For technical scheme mentioned above, in preferred situation: described condition of again fermenting is cultivate 4 ~ 7 days at 30 DEG C.
For technical scheme mentioned above, in preferred situation: described bacillus subtilis (Bacillus subtilis) CGMCC1.3382 zymotic fluid adopts nutrient medium fermentation standby; Actinomycetes (Dactylosporangium sp.) ACCCNo.40661 zymotic fluid adopts yeast extract malt extract medium fermentation standby; It is standby that rose yellow streptomycete (Streptomycesroseoflavus) ACCC No.40400 zymotic fluid employing Gao Shi synthesizes a medium fermentation; Wherein:
Nutrient medium is peptone 5g, NaCl5g, beef extract 3g, and distilled water is settled to 1.0L;
Yeast extract malt extract medium is for being yeast extract 10.0g, glucose 4g, malt extract 10.0g, and distilled water is settled to 1.0L;
It is soluble starch 20.0g, KNO that Gao Shi synthesizes a medium
31.0g, FeSO
40.01g, NaCl0.5g, MgSO
40.5g, distilled water is settled to 1.0L.
By the composition of response phase method determination final products in the embodiment of the present invention, measure the bacteriostasis of zymotic fluid finally by double-layer agar technique.Through verifying on the spot, rationally, stable effect, the pathogen of the efficient antagonism deep scab of potato of energy, increase yield, improve utilization rate of fertilizer to its formula, improvement ecological environment of soil.
Embodiment
Microbial bacterial agent of the present invention adopts bacillus subtilis (Bacillus subtilis) CGMCC1.3382, actinomycetes (Dactylosporangium sp.) ACCC No.40661, rose yellow streptomycete (Streptomyces roseoflavus) ACCC No.40400 all can be obtained by commercial sources.For convenience of describing hereinafter summary for bacillus subtilis, actinomycetes, rose yellow streptomycete.
Embodiment 1: the preparation of zymotic fluid
1) inoculate: the solid culture medium preparing each bacterial classification, strictly carry out sterile working after sterilizing, be forwarded to flat board from inclined-plane, cultivate 2 ~ 3 days under optimum temperature;
2) one-level is cultivated: be forwarded to by the dull and stereotyped picking list bacterium colony grown and be equipped with in the 50mL conical flask of level liquid medium, sterile working, 30 DEG C, 130r/min cultivates 24 ~ 48h;
3) second order fermentation: one-level is cultivated strain inoculation in the 500mL conical flask filling secondary liquid medium, sterile working, 30 DEG C, 130r/min cultivates 24 ~ 48h, obtained bacillus subtilis (Bacillus subtilis) CGMCC1.3382 zymotic fluid, actinomycetes (Dactylosporangium sp.) ACCC No.40661 zymotic fluid, rose yellow streptomycete (Streptomyces roseoflavus) ACCC No.40400 zymotic fluid respectively.
Medium used in said method:
The solid culture medium that described bacillus subtilis adopts is nutrient agar medium: peptone 5g, NaCl5g, beef extract 3g, agar 15g, and distilled water is settled to 1.0L; Level liquid medium and secondary liquid medium are nutrient medium: peptone 5g, NaCl5g, and beef extract 3g, distilled water is settled to 1.0L.
The solid culture medium that described actinomycetes adopt is yeast extract malt extract agar medium: yeast extract 10.0g, glucose 4g, malt extract 10.0g, agar 20.0g, and distilled water is settled to 1.0L; Level liquid medium and secondary liquid medium are yeast extract malt extract medium: yeast extract 10.0g, glucose 4g, malt extract 10.0g, and distilled water is settled to 1.0L;
The solid culture medium that described rose yellow streptomycete adopts is that Gao Shi synthesizes an agar medium: soluble starch 20.0g, KNO
31.0g, FeSO
40.01g, NaCl0.5g, MgSO
40.5g, agar 20g, distilled water is settled to 1.0L; Level liquid medium and secondary liquid medium are Gao Shi and synthesize a medium: soluble starch 20.0g, KNO
31.0g, FeSO
40.01g, NaCl0.5g, MgSO
40.5g, distilled water is settled to 1.0L.
Embodiment 2: the optimization of fermentation strain condition of culture
Embodiment 1 is cultivated to bacillus subtilis (Bacillus subtilis) the CGMCC1.3382 zymotic fluid obtained, actinomycetes (Dactylosporangium sp.) ACCC No.40661 zymotic fluid, rose yellow streptomycete (Streptomycesroseoflavus) ACCC No.40400 zymotic fluid is respectively by 30 ~ 35%, 30 ~ 35%, the mass percent mixing of 33 ~ 38%, being inoculated in by mixed bacterium liquid fills in the fermentation tank of fermentation medium, inoculum concentration is 1 ~ 5% of total fermentation medium quality, cultivate 4 ~ 7 days at 30 DEG C, bacterium number in zymotic fluid is made to reach 10 × 10
8individual/more than mL, namely obtains the microbial bacterial agent of described antagonism deep scab of potato.Wherein, described fermentation medium is: glucose 15 ~ 30g/L, starch 10 ~ 15g/L, peptone 15 ~ 23g/L, NaCl5 ~ 10g/L, KH
2pO
40.1 ~ 0.5g/L, all the other are distilled water; PH6.0 ~ 8.0.
1. the suitableeest carbon source, 2. the suitableeest carbon source concentration, 3. the suitableeest nitrogenous source, 4. the suitableeest nitrogen concentration for fermentation medium used in embodiment 1 carry out following optimization experiment; Respectively following optimization experiment is carried out to the 5. optimum temperature of fermentation culture conditions, 6. optimal pH, 7. the suitableeest inoculum concentration simultaneously; Eventually through Responds Surface Methodology determination optimal conditions of fermentation:
1. the suitableeest carbon source: replace in medium glucose with glucose, sucrose, lactose, maltose, fructose, wood sugar and the soluble starch of 1% respectively.Cultivate a period of time, within every 24 hours, get a culture fluid, use spectrophotometric determination amount of growth.Make Duplicate Samples.Determine with glucose and soluble starch for the suitableeest carbon source.
2. the suitableeest carbon source concentration: it is constant to fix other conditions, the suitableeest carbon source of research variable concentrations is on the impact of strain growth.The carbon source selecting initial concentration to be respectively 0.25%, 0.5%, 0.75%, 1%, 1.25%, 1.5%, 2% is studied.Cultivate a period of time, within every 24 hours, get a culture fluid, use spectrophotometric determination amount of growth.Make Duplicate Samples.Determine glucose 2%, soluble starch 1%.
3. the suitableeest nitrogenous source: use the peptone of 1%, dusty yeast, tryptone, beef extract, ammonium phosphate, ammonium nitrate, urea as the nitrogenous source of fermentation medium respectively.Cultivate a period of time, within every 24 hours, get a culture fluid, use spectrophotometric determination amount of growth.Make Duplicate Samples.Determine that with peptone be the suitableeest nitrogenous source.
4. the suitableeest nitrogen concentration: it is constant to fix other conditions, the suitableeest carbon source of research variable concentrations is studied the nitrogenous source that the impact of strain growth selects initial concentration to be respectively 0.25%, 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 2.0%.Cultivate a period of time, within every 24 hours, get a culture fluid, use spectrophotometric determination amount of growth.Make Duplicate Samples.Determine with peptone 1.5%.
5. optimum temperature: prepare medium on the basis that above condition is determined, after autoclaving by 5% inoculum concentration access seed liquor, be placed on 20 DEG C, 25 DEG C respectively, 30 DEG C, 35 DEG C, shaking table in cultivate.Within every 24 hours, get a culture fluid, use spectrophotometric determination amount of growth.Make Duplicate Samples.Determine that the suitableeest cultivation temperature is 30 DEG C.
6. optimal pH: on the basis that above condition is determined, a series of initial pH medium of preparation pH6-11, by 5% inoculum concentration access seed liquor after autoclaving, optimum temperature is cultivated.Within every 24 hours, get a culture fluid, use spectrophotometric determination amount of growth.Make Duplicate Samples.Determine that optimal pH is 7.0.
7. the suitableeest inoculum concentration: under above-mentioned optimal culture condition, respectively with 1% in 10ml liquid nutrient medium, 3%, 5%, 7%, 9% inoculum concentration access seed culture fluid, shaking table is cultivated, and within every 24 hours, gets a culture fluid, uses spectrophotometric determination amount of growth.Make Duplicate Samples.Determine that the suitableeest inoculum concentration is 3%.
8. Responds Surface Methodology determination optimal conditions of fermentation is glucose 15 ~ 30g/L, starch 10 ~ 15g/L, peptone 15 ~ 23g/L, NaCl5 ~ 10g/L, KH
2pO40.1 ~ 0.5g/L, all the other are distilled water; PH6.0 ~ 8.0, inoculum concentration is cultivate 4 ~ 7 days at 1 ~ 5%, 30 DEG C.
Embodiment 3: bacterial classification compound and liquid deep layer fermenting
By each bacterial classification by following mass percent mixing namely: fermentation of bacillus subtilis liquid 30%, actinomycetes fermentation liquor 35%, rose yellow streptomycete zymotic fluid 35%.Being inoculated in by mixed bacterium liquid fills in the fermentation tank of fermentation medium, and inoculum concentration is cultivate 5 days at 3%, 30 DEG C of total fermentation medium quality, makes bacterium number in zymotic fluid reach 10 × 10
8individual/more than mL, namely obtains the microbial bacterial agent of described antagonism deep scab of potato.Wherein, described fermentation medium is: glucose 15 ~ 30g/L, starch 10 ~ 15g/L, peptone 15 ~ 23g/L, NaCl5 ~ 10g/L, KH
2pO
40.1 ~ 0.5g/L, all the other are distilled water; PH6.0 ~ 8.0.
Embodiment 4: the mensuration of bacterial classification bacteriostasis
Adopt double-layer agar technique: 1. prepare Gao Shi and synthesize an agar medium, in culture dish, add 2 ~ 3 diameters after solidifying is the filter paper of 0.6cm, and drop 10 μ L streptomycete spore suspension (20% glycerine ,-20 DEG C of preservations) on it, spore concentration is 10
7~ 10
8cuf/mL, 30 DEG C of cultivations.
2. after cultivating 3d, in culture dish, put into a glass lens, it adds 3mL chloroform, add a cover process lh, kill shot hole streptomycete thalline; Then eyeglass is shifted out together with unnecessary chloroform, dry in the air and put 30min.
3. the mensuration of zymotic fluid bacteriostasis: add 15mL fermentation agar medium again in culture dish, add 150 μ L zymotic fluids after solidifying, smoothen with glass bar.After cultivating 3d to 30 DEG C, antibacterial circle diameter reaches 5cm, has good fungistatic effect.
Embodiment 5: the microbial bacterial agent Field information effect of antagonism deep scab of potato
The microbial bacterial agent Field information test of antagonism deep scab of potato is as follows:
1. testing program and design:
Product of the present invention is used in the experimental field that deep scab of potato is serious.
Time of application is potato tubers Formation period.
Application process is soil irrigation, not use the same kind potato of the same state of an illness in the same region of this product for experimental control.
Field trial kind: Shandong potato No. 1 production potato seed.
Test site: Shandong, open field live broadcast, covering with plastic film.
2. deep scab of potato grade scale:
Wear cotton gloves and remove soil, do not damage potato block epidermis.Range estimation.0 grade: potato block is without scab; 1 grade: have 1-3 obviously scab, or have the full of stains or spots point of multiple minor illness, but overall harm is very light; 2 grades: have more than 4 obvious scabs.
3. testing result and analysis:
The microbial bacterial agent of antagonism deep scab of potato is to sick potato preventive effect
| Group number | 1 group | 2 groups | 3 groups | Mean value |
| The Disease index (%) of CK | 37 | 40 | 36 | 37.7 |
| The Disease index (%) of test | 20 | 22 | 17 | 19.7 |
| Relative control effect (%) | 46 | 45 | 52.7 | 47.9 |
Note: disease index=(0 grade × 0 grade block number+1 grade × 1 grade block number+2 grades × 2 grades block numbers)/(2 grades × total progression)
Protection effect=(the average disease index of the average disease index-process of CK)/(average disease index of CK)
In addition, potato block color of the leather light, the good luster of production of the present invention is used; The potato block color of the leather that blank group is produced is dark.
4. conclusion:
Test illustrates, the protection effect of product prevention potato scab of the present invention is 47.9%.And make potato block color of the leather light, good luster, rise appreciably.
This test soil plot system spring sowing potato continuous cropping 4 years, deep scab of potato morbidity is not especially severe.In the soil plot test that deep scab of potato is fallen ill heavier, the protection effect of product of the present invention to deep scab of potato reaches more than 90%.
Claims (7)
1. the preparation method of the microbial bacterial agent of an antagonism deep scab of potato, it is characterized in that: adopt bacillus subtilis (Bacillus subtilis) CGMCC1.3382 zymotic fluid, actinomycetes (Dactylosporangium sp.) ACCC No.40661 zymotic fluid, as mix bacterium agent after the mixing of rose yellow streptomycete (Streptomyces roseoflavus) ACCC No.40400 zymotic fluid, obtain through fermentation again, wherein said bacillus subtilis (Bacillus subtilis) CGMCC1.3382 zymotic fluid, actinomycetes (Dactylosporangium sp.) ACCC No.40661 zymotic fluid, rose yellow streptomycete (Streptomyces roseoflavus) ACCC No.40400 zymotic fluid, respective bacterial classification quantity is all not less than 10
8individual/mL, the mass percent of three's mixing is respectively 30 ~ 35%, 30 ~ 35%, 33 ~ 38%.
2. the preparation method of microbial bacterial agent according to claim 1, is characterized in that: the inoculum concentration of described mix bacterium agent through again fermenting is that mix bacterium agent accounts for 1 ~ 5% of total fermentation medium quality.
3. the preparation method of microbial bacterial agent according to claim 1, is characterized in that: the described fermentation medium again fermented is: glucose 15 ~ 30g/L, starch 10 ~ 15g/L, peptone 15 ~ 23g/L, NaCl5 ~ 10g/L, KH
2pO
40.1 ~ 0.5g/L, all the other are distilled water; PH6.0 ~ 8.0.
4. the preparation method of microbial bacterial agent according to claim 1, is characterized in that: the end-product total bacteria count that described fermentation again obtains is at least 10 × 10
8individual/mL.
5. the preparation method of microbial bacterial agent according to claim 1, is characterized in that: described condition of again fermenting is cultivate 4 ~ 7 days at 30 DEG C.
6. the preparation method of microbial bacterial agent according to claim 1, is characterized in that: described bacillus subtilis (Bacillus subtilis) CGMCC1.3382 zymotic fluid adopts nutrient medium fermentation standby; Actinomycetes (Dactylosporangium sp.) ACCC No.40661 zymotic fluid adopts yeast extract malt extract medium fermentation standby; It is standby that rose yellow streptomycete (Streptomyces roseoflavus) ACCC No.40400 zymotic fluid employing Gao Shi synthesizes a medium fermentation.
7. the preparation method of microbial bacterial agent according to claim 6, is characterized in that: described nutrient medium is peptone 5g, NaCl5g, beef extract 3g, and distilled water is settled to 1.0L; Yeast extract malt extract medium is for being yeast extract 10.0g, glucose 4g, malt extract 10.0g, and distilled water is settled to 1.0L; It is soluble starch 20.0g, KNO that Gao Shi synthesizes a medium
31.0g, FeSO
40.01g, NaCl0.5g, MgSO
40.5g, distilled water is settled to 1.0L.
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| CN201310665843.9A CN104686582A (en) | 2013-12-06 | 2013-12-06 | Preparation method of microorganism bacterium agent for resisting potato scab |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106804627A (en) * | 2017-02-07 | 2017-06-09 | 北京国拓生物科技有限公司 | A kind of complex microorganism Wettable pesticide powder and preparation method thereof and purposes |
| CN108130302A (en) * | 2018-01-31 | 2018-06-08 | 深圳市芭田生态工程股份有限公司 | A kind of special disease-resistant microbial inoculum of potato and preparation method thereof |
| CN113373092A (en) * | 2021-06-30 | 2021-09-10 | 海南金雨丰生物工程有限公司 | Compound microbial preparation and preparation method thereof |
-
2013
- 2013-12-06 CN CN201310665843.9A patent/CN104686582A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106804627A (en) * | 2017-02-07 | 2017-06-09 | 北京国拓生物科技有限公司 | A kind of complex microorganism Wettable pesticide powder and preparation method thereof and purposes |
| CN106804627B (en) * | 2017-02-07 | 2019-06-18 | 北京国拓生物科技有限公司 | A kind of complex microorganism Wettable pesticide powder and preparation method thereof and purposes |
| CN108130302A (en) * | 2018-01-31 | 2018-06-08 | 深圳市芭田生态工程股份有限公司 | A kind of special disease-resistant microbial inoculum of potato and preparation method thereof |
| CN108130302B (en) * | 2018-01-31 | 2021-05-07 | 深圳市芭田生态工程股份有限公司 | Special anti-disease microbial agent for potatoes and preparation method thereof |
| CN113373092A (en) * | 2021-06-30 | 2021-09-10 | 海南金雨丰生物工程有限公司 | Compound microbial preparation and preparation method thereof |
| CN113373092B (en) * | 2021-06-30 | 2023-09-15 | 海南金雨丰生物工程有限公司 | Composite microbial preparation and preparation method thereof |
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Application publication date: 20150610 |