CN100366728C - Bacterial strain for preventing and treating cotton verticillium wilt and its preparing method - Google Patents
Bacterial strain for preventing and treating cotton verticillium wilt and its preparing method Download PDFInfo
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- CN100366728C CN100366728C CNB2003101096198A CN200310109619A CN100366728C CN 100366728 C CN100366728 C CN 100366728C CN B2003101096198 A CNB2003101096198 A CN B2003101096198A CN 200310109619 A CN200310109619 A CN 200310109619A CN 100366728 C CN100366728 C CN 100366728C
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- verticillium wilt
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Abstract
The present invention relates to a bacterial strain for preventing and controlling cotton verticillium wilt and a preparation method of a bacterial agent of the bacterial strain. The bacterial strain is a bacillus subtilis NCD-2 strain CGMCC1019. The preparation method of the bacterial agent of the bacterial strain adopts a three stage fermentation technology, and the inoculated bacterial strain is bacillus subtilis NCD-2; the temperature of liquid fermentation is controlled at 28 to 37 DEG C, and the initial pH value is controlled at 7.0 to 8.0. On the basis of thallus liquid fermentation, various additives are compounded to prepare a liquid preparation, or a fermentation liquor is concentrated to obtain spores; then, the liquid preparation or the spores are mixed with solid fillers according to the proportion of 1: 1000; subsequently, a solid preparation is prepared through baking and pulverization. The present invention has the advantages of obvious prevention and control effect (56.3% to 92.6%) on preventing and controlling cotton verticillium wilt, no pollution, no harm, low cost and convenience for large-area popularization and application.
Description
Technical field
The present invention relates to a kind of preparation method who prevents and treats the bacterial strain and the microbial inoculum thereof of cotton verticillium wilt, belong to the technical field of utilizing microbial control soil to pass Plant diseases.
Background technology
Cotton verticillium wilt is an of paramount importance worldwide refractory disease on the cotton that is caused by big beautiful Verticillium (Verticillium dahliae Kleb.), does not still have the effectively preventing measure at present.Statistics shows that the annual cotton verticillium wilt onset area of China accounts for 50% of sown area, and nearly 2,000,000 loads of year loss are amounted to 1,000,000,000 yuan of Renminbi, have had a strong impact on the development of Cotton Production and textile industry.Therefore, the harm of control cotton verticillium wilt oneself become and produce to go up urgent problem.
The cotton Huang bacterium that withers is typical soil inhabitant, and its control is extremely difficult.Why so this bacterium wildness causes harm and is difficult to control, its major cause is that it can be on the crop invalid body, even directly survive for the more than ten years in the soil midium or long term, and the bacterium amount can accumulate throughout the year, the present method of preventing and treating, comprise and use disease-resistant variety, chemical agent and agricultural measures, all can not reach the ideal protection effect.In default of the antigen breeding material, the verticillium wilt resistance of cotton by same breed breeding is restricted; Chemical agent prevents and treats that preventive effect is not good enough first, and second contaminate environment is destroyed the agricultural ecological balance; Crop rotation for many years comparatively effectively in the agricultural measures, but under the specific agricultural environment of China, have a large population and a few land and crop species single, be difficult to apply.
Utilize the microbial control Plant diseases particularly soil to pass Plant diseases be one of effectively preventing measure, at present, by retrieval, find to utilize the report of subtilis (B.subtilis) control cotton verticillium wilt as yet.
Summary of the invention
Technical problem to be solved by this invention provides a kind of preparation method who prevents and treats the bacterial strain and the microbial inoculum thereof of cotton verticillium wilt.
The technical solution adopted for the present invention to solve the technical problems:
One, the bacterial strain of control cotton verticillium wilt:
The NCD-2 bacterial strain is accredited as bacillus (Bacillus) subtilis (Bacillus subtilis) through the API system.
Preservation date: on October 15th, 2003, depositary institution's title and abbreviation: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC), preserving number: 1019.
Two, the preparation method of the microbial inoculum of control cotton verticillium wilt:
This method comprises that from original seed the three grade fermemtation technology through triangular flask, seeding tank, fermentor tank is characterized in that:
(1) bacterial strain that is used to inoculate is subtilis NCD-2;
(2) leavening temperature, pH value and put a jar condition:
A. the temperature of liquid fermenting is controlled at 28-37 ℃, preferred 30-32 ℃; Initial p H value is controlled at 7.0-8.0, preferred 7.2-7.6;
B. put a jar condition: microscopy observes that the gemma production rate is more than 90% in the fermented liquid, and cell concentration reaches 10
10More than;
Fermentation time is generally relevant with the volume of the composition of substratum, leavening temperature, fermentor tank, aeration condition etc.Usually the incubation time that shakes bottle and seeding tank is 1.5-2 days, and the fermentation time of fermentor tank is 2-4 days, and total fermentation time is about a week.
(3) above-mentioned fermented liquid is made liquid preparation or solid preparation:
A. the preparation of liquid preparation:
With fermented liquid and following additive by the compound liquid preparation of making of ratio requirement:
Chitin 0.3%-0.5%Kg/L (fermentating liquid volume)
Collagen protein 0.2%-0.5%Kg/L (fermentating liquid volume)
Methylcellulose gum 0.5%-1%Kg/L (fermentating liquid volume)
B. the preparation of solid preparation:
Fermented liquid concentrate is obtained gemma, again with solid filler according to 1: 1000 mixed of ratio of weight and number, solid preparation is made in drying and pulverizing then.
Described solid filler is mixed by following weight percent by wilkinite and glucose: wilkinite 99.7%, glucose 0.3%.
The weight percent of each component of seed tank culture base is:
Semen Maydis powder 2.0%
Soybean cake powder 2.0%
Wheat bran 2.0%
Na
2HPO
4 0.2％
NaH
2PO
4 0.02％
MgSO
4 0.03％
ZnSO
4 0.05％
α-Dian Fenmei 3-5u/kg batching
Bubble enemy 0.02%
The weight percent of fermentation tank culture medium each component is:
Semen Maydis powder 2.0%
Soybean cake powder 2.0%
Wheat bran 2.0%
Na
2HPO
4 0.2％
NaH
2PO
4 0.02％
MgSO
4 0.03％
ZnSO
4 0.05％
CaCO
3 0.3％
α-Dian Fenmei 3-5u/kg batching
Bubble enemy 0.02%
The method for screening and separating and the process of withered grass bud bacillus NCD-2 bacterial classification:
Lack at cotton verticillium wilt under the background of prophylactico-therapeutic measures, the inventor's at first proposition in the world " germ nuclear border " notion, and carry out germ nuclear border microorganism and separate and antagonistic microbe screening and application.The inventor used general soil bacterium separate mode in 1997; bacterium in 20 years cotton field soil of the continuous cropping of Inst. of Plant Protection, Hebei-Prov. Academy of Agricultural and Forestry Scie is separated; a bacterial isolates surplus being separated to 2000; find that NCD-2 bacterial strain and other 18 bacterial isolateses have stronger restraining effect to germ nuclear and the mycelial growth of big beautiful Verticillium after using its effect of face-off cultural method rough determination to big beautiful Verticillium (V.dahliae) mycelial growth.The cultural method of application face-off subsequently studies in great detail the bacteriostatic action of NCD-2 bacterial strain, proves that NCD-2 bacterial strain bacteriostasis rate is 57.4%, produces tangible antibacterial band simultaneously and reaches 7.0 millimeters.Indoor by the potted plant usefulness NCD-2 bacterial strain suspension (10 that experiment showed,
8Thalline/ml) to cotton seeds seed soaking handle the disease index that can the utmost point reduces cotton verticillium wilt significantly, protection effect reaches 77.0%.Illustrate that the NCD-2 bacterial strain is a bacterial strain that potentiality to be exploited is arranged very much, field plot trial was set in 1999, test method is as follows: (cfu is 10 with NCD-2 bacterial strain suspension
10Bacterium/ml), according to 2: 1 ratio (bacterium liquid: the Drug coating volume) make Drug coating with Drug coating A thorough mixing.Drug coating with grain weight 10% (volume/weight) carries out dressing to cotton seeds.The cotton seeds of dressing is broadcast in the nutrition pot, and every alms bowl is broadcast 3 seeds, blinding and compacting gently.Normal sowing time shift to an earlier date 10 day than local cotton sowing time, and nutrition pot after planting is placed in the plastic canopy, after the week of emerging, stays a strong sprout in every alms bowl.When cotton seedling has 2 true leaves, be transplanted to the land for growing field crops, during transplanting, pour into 20ml antagonistic bacterium suspension (antagonistic bacterium live body concentration 10 in every cave
7Bacterium/ml).With the clear water coating seeds, pour into clear water in the cave and be contrast.Every sub-district 15m
2, repeat random alignment 3 times.Plot experiment result shows that the processing of NCD-2 bacterial strain can prevent and treat cotton verticillium wilt, and protection effect reaches 78.1%, thereby has established the biocontrol strain of NCD-2 bacterial strain as special control cotton verticillium wilt.Subsequently, 2000-2002, respectively in Hebei province, Shandong Province and Xinjiang carried out the multipoint cell test, mainly uses NCD-2 bacterial strain solid preparation, using method is normally sowed after evenly dressing seed by 10% of grain weight, and protection effect reaches 56.3%-92.6%.
Strain liquid preparation using method of the present invention is as follows: dress seed, grow seedlings by 20% of grain weight (lint seed), 130 times of water liquid using said preparation when transplanting dip in root.
Bacterial strain solid preparation using method of the present invention is as follows: normally sow after evenly dressing seed by 10% of grain weight again.
Subtilis NCD-2 bacterial strain provided by the invention not only has had strong inhibitory effects to Huang bacterium and germ thereof the nuclear that withers through biological assay, and to cotton thorn dish spore bacterium (cotton anthracnose bacterium), Fusarium oxysporum Schl.f.sp.vasinfectum (cotton-wilt fusarium), the protruding navel of the big spot of the corn spore bacterium (Exserohilum turcicum) that wriggles, Zea mays Bipolaris sacchari (southern corn leaf blight), curvularia lunata (corn is bent the mould leaf spot fungi of spore), fusarium moniliforme (the rotten bacterium of corn stem foot), cereal rhizoctonia (rhizoctonia cerealis), Fusarium graminearum (fusarium graminearum), standing grain is given birth to anthrax-bacilus (jowar anthrax bacteria), the wheel big stem point bacterium of line (ring rot of apple bacterium), chain lattice spore apple specialized form (alternaria leaf spot of apple bacterium), white rot shield shell mould (fruit white rot of grape bacterium), alternaria solani sorauer (tomato early blight bacterium) and green onion chain lattice spore 14 kinds of staple crops pathogenic bacterias such as (purple blotch of onion bacterium) have bacteriostatic action, illustrate that antagonistic bacterium NCD-2 has the broad spectrum antimicrobial spectrum.The biological safety measurement result shows that NCD-2 is nonpathogenic to present domestic essential species plant (wheat, corn, cotton, potato, cucumber, eggplant and soybean).In addition,, and add the preparation of this bacterial strain of Additive Production, then be more convenient for large scale application if subtilis NCD-2 of the present invention is adopted the three grade fermemtation technology of liquid fermenting.
Beneficial effect of the present invention is used to prevent and treat cotton verticillium wilt with microbial inoculum of the present invention, and prevention effect is remarkable, and prevention effect reaches 56.3%-92.6%, and pollution-free, nuisanceless, low-cost, be convenient to large scale application.
Subtilis (Bacillus subtilis) NCD-2 bacterial strain:
Preservation date: on October 15th, 2003
Depositary institution's title and abbreviation: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC)
Preserving number: 1019
Depositary institution address: north, Zhong Guan-cun, Haidian District, BeiJing, China city No. 13 Institute of Microorganism, Academia Sinica of a street
Embodiment
Embodiment: the preparation method of subtilis NCD-2 bacterial strain preparation:
In strict accordance with operating about the processing step among the control cotton verticillium wilt microbial inoculum preparation method in the foregoing invention content part, can produce the liquid or solid preparation of this bacterial strain.
Claims (5)
1. a bacterial strain of preventing and treating cotton verticillium wilt is characterized in that this bacterial strain is subtilis (Bacillus subtilis) NCD-2 bacterial strain CGMCC1019.
2. preparation method who prevents and treats the microbial inoculum of cotton verticillium wilt, it includes from original seed, and the three grade fermemtation technology through triangular flask, seeding tank, fermentor tank is characterized in that:
The bacterial strain that is used to inoculate is bacterial strain CGMCC1019;
(1) leavening temperature, pH value and put a jar condition:
A. the temperature of liquid fermenting is controlled at 28-37 ℃, and initial p H value is controlled at 7.0-8.0;
B. put a jar condition: microscopy observes that the gemma production rate is more than 90% in the fermented liquid, and cell concentration reaches 10
10More than;
(2) above-mentioned fermented liquid is made liquid preparation or solid preparation:
A. the preparation of liquid preparation:
With fermented liquid and following additive by the compound liquid preparation of making of ratio requirement: chitin 0.3%-0.5% Kg/L is that chitinous weight/fermentating liquid volume collagen protein 0.2%-0.5% Kg/L is that the weight/fermentating liquid volume methylcellulose gum 0.5%-1% Kg/L of collagen protein is the weight/fermentating liquid volume of methylcellulose gum
B. the preparation of solid preparation:
Fermented liquid concentrate is obtained gemma, again with solid filler according to 1: 1000 mixed of ratio of weight and number, solid preparation is made in drying and pulverizing then.
3. the preparation method of the microbial inoculum of control cotton verticillium wilt according to claim 2 is characterized in that described solid filler is mixed by following weight percent by wilkinite and glucose: wilkinite 99.7%, glucose 0.3%.
4. the preparation method of the microbial inoculum of control cotton verticillium wilt according to claim 3 is characterized in that the weight percent of each component of seed tank culture base is:
Semen Maydis powder 2.0%
Soybean cake powder 2.0%
Wheat bran 2.0%
Na
2HPO
4 0.2％
NaH
2PO
4 0.02％
MgSO
4 0.03％
ZnSO
4 0.05％
The above-mentioned batching of α-Dian Fenmei 3-5u/kg
Bubble enemy 0.02%.
5. the preparation method of the microbial inoculum of control cotton verticillium wilt according to claim 4 is characterized in that the weight percent of fermentation tank culture medium each component is:
Semen Maydis powder 2.0%
Soybean cake powder 2.0%
Wheat bran 2.0%
Na
2HPO
4 0.2％
NaH
2PO
4 0.02％
MgSO
4 0.03％
ZnSO
4 0.05％H
CaCO
3 0.3％
The above-mentioned batching of α-Dian Fenmei 3-5u/kg
Bubble enemy 0.02%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101096198A CN100366728C (en) | 2003-10-17 | 2003-10-17 | Bacterial strain for preventing and treating cotton verticillium wilt and its preparing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2003101096198A CN100366728C (en) | 2003-10-17 | 2003-10-17 | Bacterial strain for preventing and treating cotton verticillium wilt and its preparing method |
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|---|---|
| CN1528885A CN1528885A (en) | 2004-09-15 |
| CN100366728C true CN100366728C (en) | 2008-02-06 |
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Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101643710B (en) * | 2009-08-25 | 2011-05-04 | 山东泰丰源生物科技有限公司 | Bacillus subtilis and application thereof |
| CN101671633B (en) * | 2009-09-28 | 2010-11-10 | 南京农业大学 | Antagonistic bacteria for preventing and eliminating greensickness of continuous cropping cotton and microbial organic fertilizer thereof |
| CN102515954A (en) * | 2011-12-16 | 2012-06-27 | 上海绿乐生物科技有限公司 | Special biological organic fertilizer for verticillium wilt and production method for same |
| CN102732444B (en) * | 2012-02-09 | 2013-06-19 | 河北省农林科学院植物保护研究所 | Bacillus pumilus for controlling cottonbollblight and microorganism bacterium agent thereof |
| CN103667099B (en) * | 2013-08-13 | 2015-08-26 | 浙江大学 | A kind of subtilis for preventing and treating crop fusarium disease and application thereof |
| CN103952328B (en) * | 2013-11-13 | 2016-08-17 | 陕西省微生物研究所 | A kind of bacillus subtilis strain A33, microbial inoculum and preparation method and application |
| CN104877926B (en) * | 2014-09-01 | 2018-11-06 | 北京林业大学 | The bacterial strain of one plant of antagonism smoke tree wilt and its application |
| CN105028067B (en) * | 2015-05-11 | 2017-08-01 | 新疆康润洁环保科技股份有限公司 | A kind of method for preventing and treating verticillium wilt |
| CN106754469B (en) * | 2016-11-23 | 2019-10-11 | 湖北省农业科学院植保土肥研究所 | Endogenetic bacteria HB3S-20 and the application in cotton verticillium wilt prevention and treatment |
| CN109673671A (en) * | 2019-01-22 | 2019-04-26 | 河北省农林科学院植物保护研究所 | A kind of microorganism seed inorganic agent and preparation method for preventing and treating potato verticillium wilt |
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2003
- 2003-10-17 CN CNB2003101096198A patent/CN100366728C/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
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| 利用拮抗细菌防治棉花黄萎病. 李社增等.华中农业大学学报，第20卷第5期. 2001 * |
| 棉花黄萎病生物防治研究进展. 马平.河北农业科学，第7卷第3期. 2003 * |
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| CN1528885A (en) | 2004-09-15 |
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Granted publication date: 20080206 Termination date: 20121017 |