CN102851245A - Tobacco endophyte strain inhibiting tobacco bacterial wilt, and microbial agent thereof - Google Patents
Tobacco endophyte strain inhibiting tobacco bacterial wilt, and microbial agent thereof Download PDFInfo
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- CN102851245A CN102851245A CN201210338294XA CN201210338294A CN102851245A CN 102851245 A CN102851245 A CN 102851245A CN 201210338294X A CN201210338294X A CN 201210338294XA CN 201210338294 A CN201210338294 A CN 201210338294A CN 102851245 A CN102851245 A CN 102851245A
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Abstract
The invention discloses a tobacco endophyte strain inhibiting tobacco bacterial wilt, and a microbial agent thereof. The adopted bio-control microbe is a lysinibacillus tobaccocum Kpa strain. The strain is collected in China General Microbiological Culture Collection Center on May 29th, 2012, and has a collection number of CGMCC No. 6161. The microbe inhibits the growth of tobacco bacterial wilt germs by producing active substances, and has good colonization ability at rhizospheric parts of tobacco. The microbe is a strain with industrialized production prospect. The microbial agent is Kpa strain wettable powder. The agent has excellent controlling effect against the tobacco bacterial wilt germs. The microbial agent can be used in floating seedling substrate treatment, transplanting period and mature period. The Kpa strain wettable powder prepared with the Kpa strain is characterized by low cost, high controlling effect, no residue, and safety to human and animals. The microbial agent is a high-efficiency biological pesticide for controlling bacterial wilt germs in tobacco agricultural planting. The invention provides a bio-control microbe with good effect in bacterial wilt controlling in tobacco agricultural produciton.
Description
Technical field
The present invention relates to the microbial pesticide technical field, particularly relate to a kind of tobacco endophyte bacterial strain and microbial inoculum of anti-tobacco bacterial wilt.
Background technology
Tobacco bacterial wilt be by the withered Raul Salmonella of green grass or young crops (
Ralstonia solanacearum) soil-borne disease (Peng Xiqiao of a kind of worldwide wide-scale distribution of causing, Liu Hongyan, Luo Kuan etc. the screening of Tobacco Antagonistic Endophytic Bacteria against Ralstonia Solanacearum and preliminary evaluation. Chinese Tobacco Science, 2007(2): 38-40), this disease has the characteristics such as harm is large, distribution is wide.Tobacco bacterial wilt in China Yangtze valley and south each tobacco planting district generally occur, especially with cigarette districts such as Guangdong, Fujian, Hunan, Jiangxi for (Chen Ruitai very, Zhu Xianchao, Wang Zhifa etc. national main product cigarette is economized (district) tobacco infectious disease investigation report. Chinese Tobacco Science, 1997(4): 3-9).At present, aspect the preventing and treating of tobacco bacterial wilt, mainly adopt chemical agent, but the drug effect of control tobacco bacterial wilt medicament is still not very good, and life-time service chemical agent, cause the resistance of cause of disease constantly to strengthen, dosage constantly increases, and has a strong impact on the quality of production of ecotope and tobacco leaf.Along with the enhancing of human environmental consciousness, and the needs that develop green tobacco leaf, organic tobacco leaf, biological control has become one of leading measure of prevention and control tobacco bacterial wilt.
Adopt the method for microorganism antagonism, namely utilize pathogenic Antagonistic Fungi or its extract to prevent and treat the generation of disease, have the advantages such as efficient, pollution-free and long-lasting, in the control of Plant diseases, occupy more and more important position (Huo Qinjian, Zhang Shen, Wang Ruoyan. the tobacco bacterial wilt progress. Chinese agronomy circular, 2007(8): 364-368).For the microbial control of tobacco bacterial wilt, the investigator is many to screen biocontrol microorganisms from the anosis soil in tobacco planting district.Li Qingfei etc. isolate 5 strains from the vega rhizosphere soil of Fujian have obvious inhibiting bacterium (Li Qingfei to tobacco bacterial wilt, Li Hongli, Wang Yan, Deng. the separation of tobacco bacterial wilt pathogenic bacteria and antagonism screening thereof. Henan Agricultural Sciences, 2006(1): 51-53), Lemessa etc. filter out 6 strains have obvious antagonistic action to tobacco bacterial wilt bacterium (LEMESSA F from Ethiopia's vega rhizosphere soil, ZELLER W.Screening rhizobacteria for biological control of ralstonia solanacearum in Ethiopia .Biological Control, 2007,42 (3): 336-344).Yet this class is that preventive effect is unstable from the greatest problem of the Antagonistic Fungi existence of soil separation at present.Major cause is: biocontrol microorganisms generally is subject to soil fungistasis in soil (Lockwood J is Fungistasis in soil. Biological Reviews L.1977., 52 (1): impact 1-36.), the germination rate of biocontrol microorganisms is very low, generally between 0-30%, seldom surpass 50%, therefore, be difficult in soil to set up and reach the required population density of control target object.This class biocontrol microorganisms has become the bottleneck problem that hinders its product large scale application at the colonization ability of rhizosphere.
Endophyte of plant (Plant endophyte) refers to those at the certain phase of its life history or all the stage moves in the various tissues of health plant and the microorganism of organ inside, comprise plant endogenesis epiphyte, endogenetic bacteria and Nei Sheng actinomycetes (Stone J K, Bacon C W. 2000. An overview of endophytic microbes:endo-phytism defined. Bacon C W, White J F Jr. Microbial Endophytes. New York:Marcel Dekker, P3-9).Although the separation pure culture of endophyte of plant is difficulty, endophyte of plant is easy to surely grow in plant materials, with respect to the external Biocontrol microorganism of plant, is having more advantage aspect surely growing.
Summary of the invention
First purpose of the present invention is to provide a kind of growth that suppresses tobacco ralstonia solanacearum by producing active substance, in tobacco rhizosphere good colonization ability is arranged, have the tobacco endophyte bacterial strain of the anti-tobacco bacterial wilt of suitability for industrialized production prospect.
Second purpose of the present invention is to provide a kind of microbial inoculum of the tobacco endophyte bacterial strain be used to cultivating anti-tobacco bacterial wilt.
First purpose of the present invention is achieved in that
A kind of tobacco endophyte bacterial strain of anti-tobacco bacterial wilt is tobacco Methionin genus bacillus
Lysinibacillus tobaccocumThe Kpa bacterial strain separates from the fresh blade of flue-cured tobacco, identifies acquisition that be preserved in China Microbial Culture Preservation Commission common micro-organisms center, preservation date is on 05 29th, 2012, preserving number: CGMCC No.6161.
Tobacco Methionin genus bacillus
Lysinibacillus tobaccocumThe culture condition of Kpa bacterial strain is: substratum is Jin Shi substratum (Kings Medium) (glucose 24.0 ~ 26.0 g, peptone 19.0 ~ 21.0 g, MgSO
47H
2O 1.4 ~ 1.6 g, KH
2PO
41.4 ~ 1.6g, distilled water 1000 ml), pH 7.0, inoculum size 2.5 ~ 3.5%, shake-flask culture 38 ~ 46 h, 34 ~ 37 ℃ of culture temperature; The biomass of Kpa bacterial strain reaches 2.00 * 10 during shake-flask culture
9Cfu/ml.
Second purpose of the present invention is achieved in that
A kind of microbial inoculum of the tobacco endophyte bacterial strain be used to cultivating anti-tobacco bacterial wilt, feature is: the viable count in the microbial inoculum is 6.0 ~ 6.5 * 10
9Cfu/g, water content are 6.0 ~ 6.5%, suspensibility is 70.0 ~ 72.0%, fineness is 99.5 ~ 99.7%, cross 400 mesh sieves, and the moistening time is 17.5 ~ 18.5 s, meets microbial pesticide national standard (GB 20287-2006).
Described microbial inoculum is Kpa bacterial strain wettable powder, and Kpa bacterial strain wettable powder is comprised of following raw material in per-cent: the former powder of Kpa microbial inoculum: 8 ~ 12%, and wooden sodium: 4 ~ 7%, sodium lauryl sulphate: 0.4 ~ 0.6%, sodium alginate: 0.8 ~ 1.2%, sucrose: 0.8 ~ 1.2%, diatomite: 81 ~ 83%.
The best storage temperature of the former powder of Kpa microbial inoculum and Kpa bacterial strain wettable powder is 3 ~ 5 ℃.
The preparation method of Kpa bacterial strain wettable powder is:
A, the Kpa bacterial strain is fermented in fermentor tank first, with ultrafiltration system fermented liquid is concentrated after the fermentation ends, obtain concentrated solution, then at concentrated solution on antistaling film, spread out and at indoor airing, pulverize with pulper at last and obtain the former powder of Kpa microbial inoculum;
B, the former powder of Kpa microbial inoculum is mixed according to the ratio of 20 ︰, 11 ︰, 1 ︰, 2 ︰, 2 ︰ 164 with wooden sodium, sodium lauryl sulphate, sodium alginate, sucrose and diatomite, stir, obtain at last Kpa bacterial strain wettable powder.
Tobacco Methionin genus bacillus
Lysinibacillus tobaccocumThe Kpa bacterial strain is a kind of endophyte of plant new species that the applicant separates from the fresh blade of flue-cured tobacco, identifies.This microorganism suppresses the growth of tobacco ralstonia solanacearum by producing active substance, and in tobacco rhizosphere good colonization ability is arranged, and is a kind of bacterial strain with suitability for industrialized production prospect.
Utilize the Kpa bacterial strain wettable powder of Kpa bacterial strain preparation that tobacco ralstonia solanacearum is had good prevention effect, can be used for levitating seed-cultivating interstitial substance and process transplanting time and ripening stage.Utilize that the Kpa bacterial strain wettable powder of Kpa bacterial strain preparation has that cost is low, preventive effect is high, noresidue, to person poultry safety's characteristics, be the high-performance bio agricultural chemicals of control ralstonia solanacearum in the tobacco agriculture plantation.
Kpa bacterial strain wettable powder can be mixed use with tobacco seedling medium when using, also can when transplanting, apply around the tobacco rhizosphere, or be watered the filling root in the ripening stage.Serious field is occured disease, and it is best that transplanting time and ripening stage are used respectively primary effect.
The present invention provides a kind of Biocontrol microorganism with better prevention effect for the control of bacterial wilt in the tobacco agriculture production.
Embodiment
Below in conjunction with embodiment the present invention is further detailed.
1. the preparation of the cultivation of Kpa bacterial strain and Kpa microbial inoculum
The cultivation of Kpa bacterial strain test tube slant seed: the Kpa inoculation to the inclined-plane of beef extract-peptone solid medium, is obtained Kpa bacterial strain inclined-plane seed 18-38 ℃ of lower cultivation after 2-5 days, and suitable culture condition is 36 ℃ of lower cultivations 2 days.
The cultivation of Kpa strain liquid seed: get a ring Kpa bacterial strain from streak culture flat board and receive YSP(sucrose nutrient agar) the nutrient solution, under 36 ℃, rotating speed 150 rpm cultivate behind 24 h as seed.
The preparation of Kpa bacterial strain wettable powder: in 50 L fermentor tanks with the Kpa bacterial strain (Kpa erythromycin resistance marker bacterial strain is provided by Yunnan University) of YSP culture medium culturing erythromycin resistance marker, press 5% inoculation liquid Kpa seed of culture volume, fermentation condition is 30 ℃, rotating speed 200 rpm, ventilation rate 0.8,42 h.With ultrafiltration system fermented liquid is concentrated into 1/5 of original volume after the fermentation ends.Concentrated solution is spread out on antistaling film after the fermentation, and at indoor airing, is ground into the former powder of Kpa microbial inoculum with pulper again.The former powder of Kpa microbial inoculum is mixed according to the ratio of 20 ︰, 11 ︰, 1 ︰, 2 ︰, 2 ︰ 164 with wooden sodium, sodium lauryl sulphate, sodium alginate, sucrose and diatomite, stir, obtain at last Kpa bacterial strain wettable powder.
With the active number (cfu/g) in the former powder of Kpa microbial inoculum of the dilution plate coating method Units of Account weight of routine.The former powder of 5 gKpa microbial inoculums got behind 120 ℃ of lower baking 1 min weigh the water content of the former powder of calculating K pa microbial inoculum.
Water content (%)=(initial weight-oven-dry weight) ÷ initial weight * 100%.
2. Kpa bacterial strain wettable powder is prevented and treated the greenhouse test of bacterial wilt
For trying microbial inoculum: viable count is 1 * 10
9The Kpa wettable powder of individual/g.
Tobacco bred: K326.
For trying the cause of disease material: tobacco ralstonia solanacearum.
Test method:
During the conventional floating seedlings of flue-cured tobacco, 50 g Kpa wetting properties microbial inoculums are educated the matrix mixing with the dilution of 300 ml water is rear with 1 kg; Positive control is treated to blue or green withered clever wettable powder, and blue or green withered clever 1 g educates the matrix mixing with the dilution of 300 ml water is rear with 1 kg; Blank is that 300 ml water and 1 kg educate the matrix mixing.When treating that the cigarette seedling grows to 5-6 sheet leaf, diameter be 15 cm, 500 g soil are housed, and (sandy soil: each flowerpot of vegetable mould=1:1) is transplanted a strain cigarette seedling, at cigarette root inoculation Stalk Rot 5 ml, three repetitions of every processing, every repetition 5 basins.Rich water quality management carries out according to a conventional method, and the sickness rate of each tobacco bacterial wilt of processing was added up in growth in 90 days afterwards in the room temperature greenhouse, calculated as follows sickness rate and preventive effect:
Sickness rate=morbidity strain number/investigation strain number * 100%
The sickness rate of the sickness rate-chemicals treatment of preventive effect (%)=blank
Test-results: the sickness rate of blank is 87.5%; The sickness rate that blue or green withered clever wetting properties microbial inoculum is processed is 10.8%, and preventive effect is 76.7%; The sickness rate that Kpa wetting properties microbial inoculum is processed is 11.3%, and preventive effect is 76.2%, and is suitable with the prevention effect of the blue or green withered clever wettable powder of chemical pesticide.
Claims (6)
1. the tobacco endophyte bacterial strain of an anti-tobacco bacterial wilt is characterized in that being tobacco Methionin genus bacillus
Lysinibacillus tobaccocumThe Kpa bacterial strain separates from the fresh blade of flue-cured tobacco, identifies acquisition that be preserved in China Microbial Culture Preservation Commission common micro-organisms center, preservation date is on 05 29th, 2012, preserving number: CGMCC No.6161.
2. tobacco endophyte bacterial strain according to claim 1 is characterized in that: tobacco Methionin genus bacillus
Lysinibacillus tobaccocumThe culture condition of Kpa bacterial strain is: substratum is the Jin Shi substratum, and pH 7.0, inoculum size 2.5 ~ 3.5%, shake-flask culture 38 ~ 46 h, 34 ~ 37 ℃ of culture temperature; The biomass of Kpa bacterial strain reaches 2.00 * 10 during shake-flask culture
9Cfu/ml; In the Jin Shi substratum: glucose 24.0 ~ 26.0 g, peptone 19.0 ~ 21.0 g, MgSO
47H
2O 1.4 ~ 1.6 g, KH
2PO
41.4 ~ 1.6g, distilled water 1000 ml.
3. microbial inoculum that is used for cultivating the tobacco endophyte bacterial strain of anti-tobacco bacterial wilt, it is characterized in that: the viable count in the microbial inoculum is 6.0 ~ 6.5 * 10
9Cfu/g, water content are 6.0 ~ 6.5%, suspensibility is 70.0 ~ 72.0%, fineness is 99.5 ~ 99.7%, cross 400 mesh sieves, and the moistening time is 17.5 ~ 18.5 s, meets the microbial pesticide national standard: GB 20287-2006.
4. microbial inoculum according to claim 4, it is characterized in that: described microbial inoculum is Kpa bacterial strain wettable powder, Kpa bacterial strain wettable powder is comprised of following raw material in per-cent: the former powder of Kpa microbial inoculum: 8 ~ 12%, wood sodium: 4 ~ 7%, sodium lauryl sulphate: 0.4 ~ 0.6%, sodium alginate: 0.8 ~ 1.2%, sucrose: 0.8 ~ 1.2%, diatomite: 81 ~ 83%.
5. microbial inoculum according to claim 4, it is characterized in that: the best storage temperature of the former powder of Kpa microbial inoculum and Kpa bacterial strain wettable powder is 3 ~ 5 ℃.
6. microbial inoculum according to claim 4, it is characterized in that: the preparation method of Kpa bacterial strain wettable powder is:
A, the Kpa bacterial strain is fermented in fermentor tank first, with ultrafiltration system fermented liquid is concentrated after the fermentation ends, obtain concentrated solution, then at concentrated solution on antistaling film, spread out and at indoor airing, pulverize with pulper at last and obtain the former powder of Kpa microbial inoculum;
B, the former powder of Kpa microbial inoculum is mixed according to the ratio of 20 ︰, 11 ︰, 1 ︰, 2 ︰, 2 ︰ 164 with wooden sodium, sodium lauryl sulphate, sodium alginate, sucrose and diatomite, stir, obtain at last Kpa bacterial strain wettable powder.
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Cited By (6)
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| CN105309246A (en) * | 2015-11-17 | 2016-02-10 | 中国烟草总公司郑州烟草研究院 | Root inoculation method for tobacco ingrowth biocontrol bacteria and tobacco disease and pest control method |
| CN108676741A (en) * | 2018-04-26 | 2018-10-19 | 南京农业大学 | A kind of functionality complex microorganism seedling medium and the preparation method and application thereof |
| CN110384247A (en) * | 2019-08-07 | 2019-10-29 | 云南省烟草公司曲靖市公司 | Tobacco leaf goes rotten sick biological control method in a kind of Tobacco Leaf Curing |
| CN113502250A (en) * | 2021-08-02 | 2021-10-15 | 云南省烟草农业科学研究院 | Ralstonia strain and application, acquisition and control effect evaluation method thereof |
| CN114854627A (en) * | 2022-04-29 | 2022-08-05 | 重庆西农植物保护科技开发有限公司 | Pseudomonas fluorescens for preventing and treating bacterial wilt and application thereof |
| CN114940962A (en) * | 2022-07-01 | 2022-08-26 | 中国烟草总公司四川省公司 | Tobacco seed endophyte with growth promoting effect and application thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105309246A (en) * | 2015-11-17 | 2016-02-10 | 中国烟草总公司郑州烟草研究院 | Root inoculation method for tobacco ingrowth biocontrol bacteria and tobacco disease and pest control method |
| CN108676741A (en) * | 2018-04-26 | 2018-10-19 | 南京农业大学 | A kind of functionality complex microorganism seedling medium and the preparation method and application thereof |
| CN108676741B (en) * | 2018-04-26 | 2022-04-22 | 南京农业大学 | Functional composite microorganism seedling culture substrate and preparation method and application thereof |
| CN110384247A (en) * | 2019-08-07 | 2019-10-29 | 云南省烟草公司曲靖市公司 | Tobacco leaf goes rotten sick biological control method in a kind of Tobacco Leaf Curing |
| CN110384247B (en) * | 2019-08-07 | 2020-12-22 | 云南省烟草公司曲靖市公司 | Biological control method for tobacco rot disease in tobacco leaf baking process |
| CN113502250A (en) * | 2021-08-02 | 2021-10-15 | 云南省烟草农业科学研究院 | Ralstonia strain and application, acquisition and control effect evaluation method thereof |
| CN113502250B (en) * | 2021-08-02 | 2022-08-23 | 云南省烟草农业科学研究院 | Ralstonia strain and application, acquisition and control effect evaluation method thereof |
| CN114854627A (en) * | 2022-04-29 | 2022-08-05 | 重庆西农植物保护科技开发有限公司 | Pseudomonas fluorescens for preventing and treating bacterial wilt and application thereof |
| CN114854627B (en) * | 2022-04-29 | 2023-10-13 | 重庆西农植物保护科技开发有限公司 | Pseudomonas fluorescens for preventing and treating bacterial wilt and application thereof |
| CN114940962A (en) * | 2022-07-01 | 2022-08-26 | 中国烟草总公司四川省公司 | Tobacco seed endophyte with growth promoting effect and application thereof |
| CN114940962B (en) * | 2022-07-01 | 2023-05-23 | 中国烟草总公司四川省公司 | Tobacco seed endophyte with growth promoting effect and application thereof |
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