CN102250814A - Bacillus subtilis and use thereof in bio-control preparation for preventing and controlling powdery mildew - Google Patents
Bacillus subtilis and use thereof in bio-control preparation for preventing and controlling powdery mildew Download PDFInfo
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Abstract
The invention discloses bacillus subtilis and use thereof in bio-control preparation for preventing and controlling powdery mildew and belongs to the field of microbial pesticides. The strain is (bacillus subtilis)FBR1 with a collection number of CGMCC No.4915. The industrial fermentation culture medium for the bacillus subtilis is prepared from 0.8 percent of corn flour, 1.5 percent of soybean flour, 0.1 percent of yeast powder, 0.5 percent of peptone, 0.8 percent of monopotassium phosphate, 0.15 percent of calcium carbonate, 0.02 percent of magnesium sulfate and 0.001 percent of defoamer, and by the steps of adjusting the pH value to 7.0, fixing volume by using water, and fermenting for 24 to 36 hours under conditions of a fermentation temperature of 28 to 35 DEG C, a ventilation volume of 0.2 to 0.4 v/v/min and a stirring speed of 100 to 150r/min. The invention also discloses a bio-control preparation which contains bacillus subtilis and can be used for preventing and controlling powdery mildew in economic crops such as melons, vegetables, tobacco and strawberry in place of spray chemical medicines without polluting the environment.
Description
Technical field
The invention belongs to the microbial pesticide field, relate generally to a bacillus subtilis and its industrial production process, the invention still further relates to a kind of prevent and treat cash crop Powdery Mildews such as melon, tobacco, vegetables, strawberry biological prevention and control agent.
Background technology
The kind of white powder germ is many, it is wide to distribute, at present the nearly kind more than 650 of definite designation in the world.The fungi that belongs to 3 genus, 6 kinds all can cause cucurbits powdery mildew.Cucurbits powdery mildew is mainly by single softgel shell powdery mildew (Podosphaera xanthii, former Sphaerotheca fuliginea by name) and two spore powdery mildew (Golovinomyces cichoracearum, former Erysiphe cichoracearum by name) causes, it is a kind of worldwide disease, all can take place under the various cultivation conditions, harm has seriously hindered melon green production throughout the year.White powder germ physiological strain is numerous, and the differentiation succession is fast, also is not quite similar with distribution on the different melon crops in different areas.Up to now, single softgel shell powdery mildew physiological strain has 11, and two spore powdery mildew physiological strains have 2.It has, and air-flow infects, the incubation period is short, infect characteristics such as frequent, popular strong again.This disease is brought serious harm to melon production, causes melon growth to weaken even death in seedling stage, causes the melon and fruit deformity breeding time, causes serious economy loss.Because of this germ is an obligate parasite, this for indoor control should disease research brought very big difficulty.
In China, powdery mildew G.cichoracearum mainly is distributed in Heilungkiang, Gansu, Qinghai, Xinjiang, Jiangsu; And P.xanthi mainly is distributed in ground such as Hebei, Inner Mongol, Liaoning, Jiangsu, Taiwan, Guangxi, Yunnan, Sichuan.At present, kind of cucurbits powdery mildew bacterium and the research report that physiological strain is identified are arranged also in some areas of China, as: the physiological strain 2 that identifies single softgel shell powdery mildew in the area, Hangzhou; Go out the physiological strain 1 and the 2F of single softgel shell powdery mildew in the Beijing area preliminary evaluation, the advantage physiological strain is 2F; Three subregion preliminary evaluation go out the physiological strain 1 and the 2F of single softgel shell powdery mildew in Hainan, and the advantage physiological strain is 2F, but also may have new physiological strain; The physiological strain that Xinjiang and area, Gaolan, Gansu identify single softgel shell powdery mildew is 1; And the area, Changchun identifies the main white powder germ that single softgel shell powdery mildew is this area.
Two kinds of powdery mildews all are strict obligate parasites, and their host range is all very wide, except that ground family crop, can also infect the various plants of other section.Two kinds of bacterium all have tangible specificity in parasitism, and many different mutation and physiological strain are arranged, and belong to powdery mildew of the same race on the non-melon crop together, differ and infect melon crop surely.
Because the physiological strain of white powder germ differentiation is serious, and easily develops immunity to drugs.Simultaneously, because the continuous rise of agricultural facility, for the more advantageous environment condition that provides is provided for the wildness of Powdery Mildew, the financial loss that this disease causes is increasing year by year.In agriculture production, people mainly adopt this disease of chemical pesticide control, as Du Pont's lucky star missible oil, alkene alcohol, sulphur deflocculant, many sulphur deflocculants, thiophanate methyl, m-tetrachlorophthalodinitrile, Fenbiqing, green grain husk, enemy dead worm Duokangling, Te Fuling, triadimefon, auspicious malicious copper, derosal, fluzilazol is little, celestial being is living, generation is high.But have now found that the white powder germ has produced resistance to benzimidazole class, organic phosphates, hydroxy pyrimidine class, the rare esters of gallic acid of methoxy propyl and phenoxyquinolines class, is difficult to reach the prevention effect of expection.
Chemical pesticide develops into the sixties in 20th century, the pollution of the high residue of " pesticide hazards " problem such as agricultural chemicals, the generation of the wildness of high disease and insect resistance, the biocenological havoc of natural enemy and agroecological environment etc. constantly exposes, and it is serious day by day, cause a sensation in the world, make the development of agricultural chemicals that turnover take place, drawn biological pesticide.
Biological pesticide is meant a class pesticide preparation that utilizes living organisms or its meta-bolites that harmful organisms such as insect, germ, weeds, nematode, muroid are prevented and treated, or by bionical synthetic pesticide preparation with specific action.Along with the development of society, the progress of science and technology and the enhancing of public's environmental consciousness; reduce and use chemical pesticide; the cry of protection environment for human survival grows to even greater heights; research and development utilize biological pesticide control diseases and pests of agronomic crop, develop into one of important subject of domestic and international plant protection scientific worker.Characteristics such as it is safe, effective, pollution-free that biological pesticide has match with the requirement of preserving the ecological environment and society develops in harmony.Therefore, the research and development of China's biological pesticide in recent years also begin to present the situation that makes new advances.Status and the effect of biological pesticide in integrated pest prevention and control seems more and more important.
Biocontrol microorganisms such as Verticillium lecanii (Verticillium lecanii), trichoderma harziarum (Truchoderma harziamum), Tilleiopsis pallescens, T.washingtonensis, enterobacter cloacae (Enterobacter cloacae), subtilis (Bacillus subtilis), bacillus firmus (B.firmus), streptomycete (Streptomycete spp), Xenorhabdus nematophilus (Xenorhabdus nematophlus) etc. all has good prevention effect to Powdery Mildew.But biocontrol strain derives from the greenhouse experimental result to the preventive effect majority of Powdery Mildew, and most bacterial strain big Tanaka in the open owing to be subjected to ultraviolet ray or other Effect of Environmental, is difficult to the prevention effect good to having of Powdery Mildew.
Melon crop occupies an important position in the common people's " vegetable basket project ".The generation of the method control Powdery Mildew by biological control is to the quality, the use that reduces chemical pesticide that improve melon crop, promote the harmonious development of agroecosystem to have great significance.
Summary of the invention
Purpose of the present invention is exactly in order to adapt to the growth requirement of Organic farming, a kind of subtilis to be provided, can effectively solving the existing in prior technology problem.
Another object of the present invention is to provide industrial preparation and the application thereof of this subtilis.
Another object of the present invention is to provide the biological prevention and control agent that contains above-mentioned subtilis.
Another object of the present invention is to provide the using method of described biological prevention and control agent.
Subtilis provided by the invention (Bacillus subtilis) FBR1 has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China; Deposit number is: CGMCC No.4915; Preservation date: on 05 27th, 2011.
Bacterial strain of the present invention is subtilis FBR1, separates in the cucumber rhizosphere of Zouping County, Shandong outlying area.The separation method of this bacterial strain is: claim rhizosphere soil 10g, carry out gradient dilution to 10-6 to 10-5 with sterilized water, get 100 μ L and be coated in that (the NA substratum is: beef extract 3g, yeast extract 1g, peptone 5g, glucose 10g, agar-agar 15g on the NA solid plate; Distilled water 1000.0ml, pH6.8-7.0.), choose mono-clonal, and carry out purifying, finally obtain this bacterial strain.The feature of this bacterial strain is as follows: (1) on the potato sucrose substratum, fast growth, 12-24 hour can cover with diameter is 9 centimetres culture dish, and is tangible concentric circles, bacterium colony is the purulence shape, at colony edge fold is arranged; (2) on the LB substratum, bacterium colony is the purulence shape, and is transparent, protruding steamed bun shape, and smooth surface is difficult for producing fold; Thalline is the bat shape, and size is 0.5-0.9 * 1.9-2.5 micron, and the flagellum end is given birth to, and has mobility; Produce gemma, give birth in the gemma, be ellipse.(3) this bacterial strain is strong at the plant leaf surface colonization ability, has the effect of sterilizing disease-preventing; (4) aerobic type, Gram-positive; Can utilize multiple monose or biglycan, hydrolyzable starch, Mierocrystalline cellulose, casein and gelatin, be lower than 5 ℃ or be higher than 55 ℃ all can not normal growth.
The invention also discloses the industrial process of subtilis (Bacillus subtilis) FBR1, fermention medium is: Semen Maydis powder 0.8%, analysis for soybean powder 1.5%, yeast powder 0.1%, peptone 0.5%, potassium primary phosphate 0.8%, lime carbonate 0.15%, sal epsom 0.02%, defoamer 0.005%, pH 7.0, water constant volume; Leavening temperature is 28-35 ℃, and air flow is 0.2v/v/min-0.4v/v/min, and stirring velocity is 100-150 rev/min, and fermentation time is 24-36 hour.
Preferably: also comprise sucrose 2.0% in the described fermention medium.
The invention also discloses the industrial process of subtilis (Bacillus subtilis) FBR1,
The invention also discloses the application of this subtilis (Bacillus subtilis) FBR1 in the control crop powdery mildew.
Preferably: described farm crop are melon, vegetables, tobacco or strawberry.
Preferably: described Powdery Mildew is microbial by single softgel shell white powder.
Preferably: described Powdery Mildew is microbial by two spore white powder.
The invention also discloses a kind of biological prevention and control agent, described biotechnological formulation contains the subtilis FBR1 that preserving number is CGMCCNo.4915.
The invention also discloses the field using method of above-mentioned biotechnological formulation, doubly with described biotechnological formulation dilution 0-1000.
Preferably: doubly with described biological prevention and control agent dilution 300-500.
Bacterial strain-the FBR1 that can be used for the cucurbits powdery mildew biological control of the present invention obtains its active-fermented broth by industrial fermentation, be used for the biological control of land for growing field crops cucurbits powdery mildew, and curative effect is good.This invention is easy to suitability for industrialized production, does not produce three industrial wastes, environmentally safe, to the significance that has of Organic farming development.
Description of drawings
Accompanying drawing 1 shows the form of thalline of the present invention at microscopically.
The preservation information of thalline of the present invention is as follows:
Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center;
Address: No. 1 institute in North Star West Road, Chaoyang District, BeiJing, China city;
Preservation date: on 05 27th, 2011;
Deposit number: CGMCC No.4915.
Embodiment
Following examples only are used for that the present invention will be further described, are not to be used for limiting the scope of the invention.
Embodiment 1
Bacterial strain activates and expands numerous:
With bacterial strain at LB solid medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0,1.5% agar) go up the line activation, and at LB liquid nutrient medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0) expands numerously, obtain ferment-seeded.
Industrial fermentation:
Substratum: Semen Maydis powder 1%, analysis for soybean powder 1.5%, yeast powder 0.1%, peptone 0.5%, potassium primary phosphate 0.8%, lime carbonate 0.15%, sal epsom 0.02%, defoamer 0.005%, sucrose 2.0%, pH 7.0; Fermentation condition: leavening temperature is that 28-35 ℃, air flow are 0.4v/v/min, and stirring velocity is 100 rev/mins, 24 hours; During the fermentation, sampling in 2 hours is 1 time at interval, measures the pH value, observes colonial morphology.Reach at 5% o'clock at the gemma number, add material once, reach at 90% o'clock, stop fermentation, and determine whether to be this biotechnological formulation, and add up the thalline number, contain 200,000,000 viable bacterias amounts in every milliliter with blood counting chamber by microscopic examination at the gemma number.The gained fermented liquid is biotechnological formulation of the present invention.
Embodiment 2
Bacterial strain activates and expands numerous:
With bacterial strain at LB solid medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0,1.5% agar) go up the line activation, and at LB liquid nutrient medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0) expands numerously, obtain ferment-seeded.
Ferment-seeded is inoculated in Semen Maydis powder 0.8%, analysis for soybean powder 1.5%, yeast powder 0.1%, peptone 0.5%, potassium primary phosphate 0.8%, lime carbonate 0.15%, sal epsom 0.02%, defoamer 0.005%, pH 7.0; Fermentation condition: leavening temperature is that 28 ℃, air flow are 0.2v/v/min, and stirring velocity is 100 rev/mins, fermentation time 36 hours; During the fermentation, sampling in 2 hours is 1 time at interval, measures the pH value, observes colonial morphology.Reach at 5% o'clock at the gemma number, add material once, reach at 90% o'clock, stop fermentation, and determine whether to be this biotechnological formulation, and add up the thalline number, contain 200,000,000 viable bacterias amounts in every milliliter with blood counting chamber by microscopic examination at the gemma number.The gained fermented liquid is biotechnological formulation of the present invention.
Embodiment 3
Bacterial strain activates and expands numerous:
With bacterial strain at LB solid medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0,1.5% agar) go up the line activation, and at LB liquid nutrient medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0) expands numerously, obtain ferment-seeded.
Ferment-seeded is inoculated in Semen Maydis powder 0.8%, analysis for soybean powder 1.5%, yeast powder 0.1%, peptone 0.5%, potassium primary phosphate 0.8%, lime carbonate 0.15%, sal epsom 0.01%, defoamer 0.005%, pH 5.5; Fermentation condition: leavening temperature is that 30 ℃, air flow are 0.2v/v/min, and stirring velocity is 100 rev/mins, 34 hours; During the fermentation, sampling in 2 hours is 1 time at interval, measures the pH value, observes colonial morphology.Reach at 5% o'clock at the gemma number, add material once, reach at 90% o'clock, stop fermentation, and determine whether to be this biotechnological formulation, and add up the thalline number, contain 200,000,000 viable bacterias amounts in every milliliter with blood counting chamber by microscopic examination at the gemma number.The gained fermented liquid is biotechnological formulation of the present invention.
Embodiment 4
Bacterial strain activates and expands numerous:
With bacterial strain at LB solid medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0,1.5% agar) go up the line activation, and at LB liquid nutrient medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0) expands numerously, obtain ferment-seeded.
Ferment-seeded is inoculated in Semen Maydis powder 0.8%, analysis for soybean powder 1.5%, yeast powder 0.1%, peptone 0.2%, potassium primary phosphate 0.8%, lime carbonate 0.15%, sal epsom 0.01%, defoamer 0.005%, pH 7.0; Fermentation condition: leavening temperature is that 35 ℃, air flow are 0.4v/v/min, and stirring velocity is 150 rev/mins, 36 hours; During the fermentation, sampling in 2 hours is 1 time at interval, measures the pH value, observes colonial morphology.Reach at 5% o'clock at the gemma number, add material once, reach at 90% o'clock, stop fermentation, and determine whether to be this biotechnological formulation, and add up the thalline number, contain 200,000,000 viable bacterias amounts in every milliliter with blood counting chamber by microscopic examination at the gemma number.The gained fermented liquid is biotechnological formulation of the present invention.
Embodiment 5
Bacterial strain activates and expands numerous:
With bacterial strain at LB solid medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0,1.5% agar) go up the line activation, and at LB liquid nutrient medium (1.0% Tryptones, 0.5% yeast extract, 1.0% sodium-chlor, pH value about 7.0) expands numerously, obtain ferment-seeded.
Ferment-seeded is inoculated in Semen Maydis powder 0.5%, analysis for soybean powder 1%, yeast powder 0.1%, peptone 0.5%, potassium primary phosphate 0.3%, lime carbonate 0.09%, sal epsom 0.02%, defoamer 0.005%, pH 8.5; Fermentation condition: leavening temperature is that 35 ℃, air flow are 0.4v/v/min, and stirring velocity is 150 rev/mins, 36 hours; During the fermentation, sampling in 2 hours is 1 time at interval, measures the pH value, observes colonial morphology.Reach at 5% o'clock at the gemma number, add material once, reach at 90% o'clock, stop fermentation, and determine whether to be this biotechnological formulation, and add up the thalline number, contain 200,000,000 viable bacterias amounts in every milliliter with blood counting chamber by microscopic examination at the gemma number.The gained fermented liquid is biotechnological formulation of the present invention.
Biological prevention and control agent of the present invention is prevented and treated the field control efficient experiment of Powdery Mildew.
1. experiment material:
With cucumber, tobacco, powdery mildew of strawberry is experimental subjects, measures the field effect of the biotechnological formulation control Powdery Mildew of FBR1 preparation.Living bacteria count is for being at least 2 * 109 in the biotechnological formulation.
2. experimental design:
100 times of A dilutions, foliage-spray; 300 times of B dilutions, foliage-spray; 500 times of C dilutions, foliage-spray; 800 times of D dilutions, foliage-spray; 1000 times of E dilutions, foliage-spray; The contrast of CK clear water.
Each processing sprays 100 plant, repeats 3 times.And sprayed once in 7 to 9 days at interval.
3. data statistics
The disease grade scale:
0 grade of no scab; 1 grade-lesion area accounts for below 1/4 of leaf area; 2 grades-lesion area accounts for the 1/4-2/4 of leaf area; 3 grades-lesion area accounts for the 2/4-3/4 of leaf area; 4 grades-lesion area accounts for more than 3/4 of leaf area.
Disease index=[∑ (sick level value * this level morbidity number of sheets)/(investigating total number of sheets * the highest sick level value)] * 100
Prevent and treat formulae of efficiency=[1-(disease index of the disease index/contrast of processing)] * 100
With the SPSS statistical software data are analyzed.
4. experimental result
Following table is the data to the powdery mildew of cucumber biocontrol effect:
Following table is the data to tobacco Powdery Mildew biocontrol effect:
Following table is the data to the powdery mildew of strawberry biocontrol effect:
From above data as can be known, on the controlling powdery mildew of three kinds of crops, 100 times of diluents (handling A) effect is best, and preventive effect is more than 90%, and highly stable; 300 times preventive effect is only second to the preventive effect of 100 times (treatments B) between 80%-90%; The prevention effect of 500 times (handling C) is about 70%; The prevention effect of 800 times (handling D) and 1000 times (handling E) is general.Consider the application cost and the prevention effect in its field, biotechnological formulation of the present invention when working concentration 300-500 times of field all can, the lasting period was at 7-9 days.And the Powdery Mildew to different cash crop all has stable prevention effect.
Claims (10)
1. a bacillus subtilis (Bacillus subtilis) FBR1 is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is: CGMCC No.4915.
2. the application of the described subtilis of claim 1 (Bacillus subtilis) FBR1 in the control crop powdery mildew.
3. application according to claim 2 is characterized in that: described farm crop are melon, vegetables, tobacco or strawberry.
4. application according to claim 2 is characterized in that: described Powdery Mildew is microbial by single softgel shell white powder.
5. application according to claim 2 is characterized in that: described Powdery Mildew is microbial by two spore white powder.
6. make the commercial run of subtilis (Bacillus subtilis) according to claim 1 for one kind, it is characterized in that: fermention medium is: Semen Maydis powder 0.5-1.0%, analysis for soybean powder 1.0-1.5%, yeast powder 0-0.1%, peptone 0.2-0.5%, potassium primary phosphate 0.3-0.8%, lime carbonate 0.09-0.15%, sal epsom 0.01-0.02%, defoamer 0.005%, pH 5.5-8.5, water constant volume; Leavening temperature is 28-35 ℃, and air flow is 0.2v/v/min-0.4v/v/min, and stirring velocity is 100-150 rev/min, and fermentation time is 24-36 hour.
7. commercial run as claimed in claim 6 is characterized in that: also comprise sucrose 0-2.0% in the described fermention medium.
8. biological prevention and control agent, it is characterized in that: described biological prevention and control agent contains the careless genus bacillus FBR1 that preserving number is CGMCC No.4915.
9. the field using method of described biological prevention and control agent according to Claim 8 is characterized in that: with described biological prevention and control agent dilution 0-1000 doubly.
10. using method according to claim 9 is characterized in that: with described biological prevention and control agent dilution 300-500 doubly.
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