CN105586298A - Bacillus subtilis, bactericide, preparing method of bactericide and application of bactericide - Google Patents
Bacillus subtilis, bactericide, preparing method of bactericide and application of bactericide Download PDFInfo
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Abstract
The invention discloses bacillus subtilis, bactericide, a preparing method of the bactericide and application of the bactericide. The bacillus subtilis is bacillus subtilis JQ-001 and is preserved in the common microorganism center of China microorganism strain preservation management committee, preservation time is November 9th, 2015, and the preservation register number is CGMCCNo.11632; the liquid bactericide or the solid bactericide can be prepared by means of seed activation, seed culture and fermentation culture. The invention further provides a preparing method of the bacillus subtilis, and application to preparation of medicine for preventing and treating rice blast and potato scab. The bactericide has high bioactivity and has the advantages of being short in production period and low in production cost; meanwhile, environment compatibility of the bactericide is good, the effects of preventing and treating rice blast and potato scab are remarkable, no hazardous effect is generated for people and livestock, and use is convenient.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to the preparation method of a kind of bacillus subtilis, microbial inoculum, microbial inoculumApplication with microbial inoculum.
Background technology
Rice blast is one of important disease of paddy rice, all has generation in Ge Dao district of the world, wherein occurs as with leaf portion, joint portionMany, after generation, can cause the underproduction in various degree, underproduction 40%-50% when serious, causes heavy loss. This disease is drawn by piricularia oryzaeRise, germ survives the winter on straw and paddy with conidium and mycelium, produces conidium next year and borrows wind and rain to propagate into rice strainUpper, sprout and invade host to adjacent cells expansion morbidity, form primary infection focus. The conidium that disease portion forms, borrows wind and rain to propagateInfect, sowing infected seed can cause seedling pest again. Under field conditions (factors), rice blast fungus only infects paddy rice, therefore to paddy riceProduce and threaten greatly.
Mainly with 500 times of liquid of 70% thiophanate methyl wettable powder, 50% carbendazim for the control of this disease at present250 times of liquid of wettable powder, 25% make 2000 times of liquid of hectogram missible oil, 500 times of liquid of strong chlorine oil wettable powder, 10% seed soaking spirit breast2500 times of liquid of oil, 3000-4000 times of liquid medicament seed soaking of 25% Shi Baoke missible oil are processed or are sprayed in the time of morbidity for 48-72 hour30% 500 times of Hinosan missible oil liquid, 50% disappear 500 times of liquid of bacterium spirit water solube powder, 500 times of liquid of 40% gram of hundred bacterium suspending agent,400 times of 25% 1500 times of Prochloraz missible oil liquid, 400 times of liquid of 40% Isoprothiolane wettable powder, 50% Rabcide wettable powderLiquid, 50% carbendazol wettable powder 500-600 times liquid, 1000 times of liquid of 70% thiophanate methyl wettable powder, 75% 3 ring2000 times of liquid of azoles wettable powder, 20% rice song overcome the enemy 1500 times of liquid of wettable powder, 400 times of liquid of 20% fentin hydroxide colloidal suspending agent etc.Chemical agent. The long-term chemical bactericide that uses, causes the germ resistance to the action of a drug to strengthen, when only having the dosage of increasing and shortening spray medicineBetween interval control disease, not only increased production cost and also caused persticide residue more and more much. A large amount of chemical drugs that useAgent, easily causes soil pollution, acid-base disorder, and nutrient content reduces, and soil compaction has a strong impact on the output of crop. Therefore adoptControl the inexorable trend that rice blast is agricultural development with biological control. To replace and adopt antagonistic microbe to prevent and treat diseaseSafe, nontoxic, the effective new method of one of chemical agent. Antagonistic microbe not only can suppress the growth of pathogen, can alsoIncrease the quantity of Soil Microorganism, Promoting plant growth, to improving the ecosystem, keeps bio-diversity, safeguards ecological flatWeighing apparatus has important effect.
Shot hole is regarded as the worldwide disease in potato production, at the many potatos of China main producing region ubiquity,Continuous cropping ground, meta-alkali ground and cultivation management improperly in situation occurrence degree even more serious. In recent years, shot hole is a lot of horses of ChinaLing Shu production area occurs in succession, wherein Heilungkiang, the Inner Mongol, Hebei, Shandong, Shanxi, Shaanxi, Guizhou, Gansu, Sichuan, lakeAll there is the report about this disease the potato main producing region of multiple provinces and regions such as south, Hubei, Yunnan, has substantially covered northeast, North China, westNorthern and southwestern. And the regional incidence of disease serious at drought, soil meta-alkalescence, continuous cropping continuous cropping is higher.
The potato scab germ stem tuber of mainly causing harm, invades from hole skin, and early stage produces moth patch at stem tuber epidermisPoint expands later gradually, infection court necrosis around, stem tuber surface roughening, quality suberification. Simultaneously according to pathogenThe difference of kind often shows as raised or sunken surperficial scab on ripe potato piece, when serious scab in flakes, the product of potato pieceMatter reduces. Meanwhile, due to epidermal tissue destroyed after, easily by other pathogen infections, cause stem tuber rot, have a strong impact on Ma LingThe outward appearance of potato, reduces its quality and storage cycle.
Utilizing biological control soil-borne disease is a kind of effective prophylactico-therapeutic measures, is in plant protection requisite groupBecome part. Particularly utilize the plant rhizosphere beneficial microbe that occurring in nature exists originally to do the antagonism of target pathogenic microorganismWith, control the harm of pathogenic microorganism, have compared with circlet border risk, be a kind of eco-friendly Prevention Technique. Along with peopleFood security, ecological safety are paid attention to, biological control is to realize one of Critical policies of agricultural modernization sustainable development.
Summary of the invention
The object of the present invention is to provide a kind of new bacillus subtilis.
Another object of the present invention is to provide described bacillus subtilis microbial agent and preparation method,
The present invention also has an object to be to provide to utilize described bacillus subtilis microbial agent at preparation controlling plant diseases medicineApplication in thing.
The object of the invention is to be achieved through the following technical solutions:
A kind of bacillus subtilis, is characterized in that: bacillus subtilis (Bacillussubtilis) JQ-001, protectsBe hidden in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: the Chaoyang District, Beijing City North StarNo. 1, West Road institute, the preservation time: on November 9th, 2015, preservation registration number is CGMCCNo.11632.
Bacillus subtilis of the present invention (Bacillussubtilis) JQ-001 is from Heilungkiang JianSanjiang farm continuous cropping 5Separation in year above paddy rice foundation soil, purifying and screening step obtain, and adopt morphological feature, Physiology and biochemistry experiment, divideSub-biological assay and 16SrDNA homologous sequence compare of analysis finally determine that the bacterial strain of above-mentioned deposit number is withered grass gemmaBacillus (Bacillussubtilis).
A kind of bacillus subtilis microbial agent is that bacillus subtilis JQ-001 is through seed activation, seed culture, fermented and culturedAfter the microbial inoculum made; This microbial inoculum comprises liquid bacterial agent and solid fungicide.
The preparation method of the microbial inoculum taking bacillus subtilis as active component, concrete steps are as follows:
A. actication of culture: the bacillus subtilis JQ-001 inoculation that low temperature is preserved is on solid medium, in 35-Cultivate 34-36h for 38 DEG C; Picking list bacterium colony is transferred on slant medium, in 35-38 DEG C of cultivation 16-24h, with under aseptic washingMedia surface thalline is as inoculation liquid;
B. primary seed solution preparation: according to the inoculum concentration of 5%-10% to accessing prepared by step a connecing in seed culture mediumPlant liquid, rotating speed 200-260rpm, temperature 35-38 DEG C, shaking table is cultivated 16-24h, as primary seed solution;
C. secondary seed solution preparation: according to the inoculum concentration of 0.5%-3% to access prepared by step b in seed culture medium oneLevel seed liquor, throughput is 0.4-1.5vvm, and mixing speed is 180-260rpm, and incubation time is 18-24h, as secondary kindSub-liquid;
The constituent of seed culture medium and percentage by weight thereof are: glucose 1%-2%, peptone 1%-1.5%, fermentFemale powder 0.6%-1%, beef extract 0.5%-0.7%, sodium chloride 0.5%, all the other are water;
D. fermented and cultured: access secondary seed prepared by step c according to the inoculum concentration of 5%-10% in fermentation mediumLiquid, throughput is 0.4-1.5vvm, and mixing speed is 180-260rpm, and incubation time is 24-30h, obtains zymotic fluid;
The constituent of fermentation medium and percentage by weight thereof are: glucose 3%-5%, peptone 0.5%-1.5%,Dusty yeast 0.03%-0.08%, peptone 0.1%-0.3%, bean cake powder 0.5%-1.5%, magnesium sulfate 0.01%-0.05%,Manganese sulfate 0.01%-0.05%, all the other are water;
E. above-mentioned zymotic fluid is concentrated by washing and molecular sieve, obtain bacillus subtilis liquid bacterial agent.
F. by above-mentioned fermented liquid spray drying: inlet temperature 150-200 DEG C, outlet temperature 50-90 DEG C, adds spraying carrierSoluble starch, obtains bacillus subtilis bacteria powder microbial inoculum.
G. following raw materials by weight is mixed: bacillus subtilis bacteria powder 20-30%; White carbon 10-15%; Diatomite: 10-20%; Dodecyl sodium sulfate 1-10%; Naphthalene sulfonate D-10022-10%; Bentonite 10-20%,Wetting agent W-20012-10%. Then utilize more than airslide disintegrating mill is crushed to 400 orders, control moisture≤5%. ObtainBacillus subtilis wettable powder microbial inoculum.
Further, the present invention also provides above bacillus subtilis microbial agent preparation control rice blast and potatoApplication in shot hole medicine.
The present invention has following beneficial effect:
The microbial inoculum being made by bacillus subtilis of the present invention is sprayed onto on crop blade face, can field planting in crop surface, account forNeck pathogen ecological niche, suppresses pathogen growth, and Pathogen of Rice Blast Fungus inhibiting rate is reached more than 91.3%; Spray application to groundOn face, by infiltration, can suppress equally pathogen growth, it reaches more than 89% potato scab pathogen inhibiting rate;And can promote plant growth, increase crop yield, maintaining ecological balance. And it is short that the production of its liquid bacterial agent has the cycle, liveThe feature that property is high, cost is low, is beneficial to suitability for industrialized production, and is prepared into solid fungicide, and being particularly prepared into can after wettable powderTo extend the preservation cycle, easy to use.
Culture presevation
Bacillus subtilis JQ-001, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center,Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation time: on November 9th, 2015, preservation registration number isCGMCCNo.11632。
Brief description of the drawings
Fig. 1 is the 16SrDNA homologous sequence compare of analysis dendrogram of bacillus subtilis of the present invention.
Detailed description of the invention
Further illustrate below in conjunction with concrete implementation column:
Embodiment mono-: the isolation and screening of bacillus subtilis JQ-001 bacterial strain:
1, separate
(1) soil collecting: from Heilungkiang JianSanjiang farm continuous cropping more than 5 years in rice terrace upper soll layer gather to 10cmSoil sample 100g, 5 samples of the three unities parallel acquisition, mixed 40 order classifying screens. Get the lower soil of 100g sieve and be used as separation.
(2) culture medium preparation: preparation respectively
NA culture medium: peptone 10g/L, beef extract 3.0g/L, sodium chloride 5g/L, agar 18g/LpH=7.0-7.2;
With PDA culture medium: potato 200g, glucose 20g, agar powder 20g, distilled water 1000mL;
Make culture medium flat plate.
(3) accurately take 1g soil and be placed in 250ml triangular flask, add 99ml sterilized water, shaking table 200rpm, 30-37 DEG CConcussion 30min. Make Soil Slurry, by 10 times of gradient dilutions to 10-8. Separate with in PDA culture medium at NA culture medium respectivelySingle bacterium colony, the single bacterium colony being separated to activates respectively 4 cycles in NA culture medium and PDA culture medium.
2, screening
It is right that the mono-bacterium colony of JQ-001 that separation is activated and other bacterial classification list bacterium colony and Pathogen of Rice Blast Fungus carry out flat boardThe experiment of standing erect is below screening process face-off experiment and result thereof:
The pathogen (rice blast) of cultivating in advance in the access of PDA culture medium flat plate central authorities, when long size to fit to be generatedGet bacterial plaque with the card punch of diameter 9mm, move to new PDA culture medium flat plate central authorities, inoculating apart from pathogen bacterial plaque 2cm placeThe existing different strain of company, each processing repeats 3 times, is placed in 25 DEG C of biochemical cultivation cases and cultivates, and observes each colony diameter, result asShown in table 1.
Table 1JQ-001 and other bacterial strain colony diameter (cm)
JQ-001 bacterial strain is the highest to the bacteriostatic activity of rice blast as can be seen from Table 1. Continue to do the short of money of JQ-001 bacterial strainAnti-experiment, method is the same, measures bacteriostasis rate. Obtain following result, in table 2.
Bacteriostasis rate (%)=(contrast colony diameter-processing colony diameter)/contrast colony diameter × 100%
Table 2JQ-001 bacterial strain is to the indoor Antibacterial Activity of rice blast
Embodiment bis-: the qualification of bacillus subtilis JQ-001 bacterial strain
1. Microbiological Characteristics: JQ-001 bacterial strain thalline is rod-short, 0.6-0.7 μ m × 1.9-3.0 μ m, single or pairedArrange, gemma column, middle life, sporangiocyst slightly expands, Gram-positive. Bacterial strain is at NA culture medium, 37 DEG C of cultivation 24h, and bacterium colony is milky whiteLook, rat, edge is irregular, dry opaque.
2. physiological and biochemical property:
The physiological and biochemical property of table 3 bacillus subtilis JQ-001 bacterial strain
| Test item | Characteristic | Test item | Characteristic | Test item | Characteristic |
| 7%Nacl growth | + | Arginine dihydrolase | - | Sweet mellow wine | + |
| 15 DEG C of growths | + | Beta galactose enzyme | - | Starch Hydrolysis | + |
| 50 DEG C of growths | + | H2S produces | - | Citric acid utilizes | + |
| D-Glucose | + | Lysine decarboxylase | + | Ornithine decarboxylase | - |
Note: "+" represents positive reaction, "-" represents negative reaction
3. molecular biology identification:
The 16SrDNA extension increasing sequence of JQ-001 bacterial strain is compared in ncbi database, same with BacillussubtilisSource property is 99.38%, in conjunction with morphological feature, the physiological and biochemical property of JQ-001 bacterial strain, determines that JQ-001 bacterial strain belongs to withered grass budSpore bacillus Bacillussubtilis, taking 16SrDNA sequence as basic JQ-001 bacterial strain phylogenetic tree is as Fig. 1 (bacterium in figurePlant 1 and be JQ-001 bacterial strain) shown in.
Embodiment tri-: the preparation of bacillus subtilis liquid bacterial agent
(1) actication of culture: by low temperature preserve bacillus subtilis JQ-001 inoculation on LB solid medium, inCultivate 36h for 36 DEG C; Picking list bacterium colony is transferred on LB slant medium, in 36 DEG C of cultivation 20h, with cultivating base table under aseptic washingFace thalline is as inoculation liquid.
(2) primary seed solution preparation: access step according to the inoculum concentration of 10% (percent by volume) in seed culture medium(1) inoculation liquid of preparing, rotating speed 220rpm, 36 DEG C of temperature, shaking table is cultivated 20h, as primary seed solution.
(3) secondary seed solution preparation: access step according to the inoculum concentration of 3% (percent by volume) in seed culture medium(2) primary seed solution of preparing, throughput is 1.0vvm, and mixing speed is 220rpm, and incubation time is 20h. As secondary kindSub-liquid.
The constituent of described seed culture medium and percentage by weight thereof are: glucose 2%, peptone 1.5%, dusty yeast1%, beef extract 0.7%, sodium chloride 0.5%, all the other are water.
(4) fermented and cultured: access step (3) preparation according to the inoculum concentration of 10% (percent by volume) in fermentation mediumSecondary seed solution, throughput is 1.0vvm, mixing speed is 220rpm, incubation time is 28h, obtains zymotic fluid.
The constituent of described fermentation medium and percentage by weight thereof are: glucose 5%, peptone 1.5%, dusty yeast0.08%, peptone 0.3%, bean cake powder 1.5%, magnesium sulfate 0.03%, manganese sulfate 0.03%, all the other are water.
(5) above-mentioned zymotic fluid is concentrated by washing and molecular sieve, obtaining viable count is the above withered grass of 1,000 hundred million/milliliterBacillus liquid microbial inoculum.
Embodiment tetra-: the preparation of bacillus subtilis bacteria powder microbial inoculum
The zymotic fluid that embodiment tri-is obtained is sprayed dry: 190 DEG C of inlet temperatures, 80 DEG C of outlet temperatures, addSpraying carrier soluble starch. Obtaining living bacteria count is 1,000 hundred million/gram above bacillus subtilis bacteria powder microbial inoculum.
Embodiment five: the preparation one of bacillus subtilis wettable powder microbial inoculum
Following raw materials by weight is mixed: bacillus subtilis bacteria powder 30% that embodiment tetra-obtains,White carbon 15%, diatomite 20%, dodecyl sodium sulfate 10%, naphthalene sulfonate D-1002:2%, bentonite 15%, wetting agentW-2001:8%. Then utilize more than airslide disintegrating mill is crushed to 400 orders, control moisture≤5%. Obtain 40,000,000,000/gramBacillus subtilis wettable powder microbial inoculum.
Embodiment six: the preparation two of bacillus subtilis wettable powder microbial inoculum
Following raw materials by weight is mixed: bacillus subtilis bacteria powder 20% that embodiment tetra-obtains,White carbon 15%, diatomite 20%, dodecyl sodium sulfate 5%, naphthalene sulfonate D-1002:10%, bentonite 20%, wetting agentW-2001:10%. Then utilize more than airslide disintegrating mill is crushed to 400 orders, control moisture≤5%. Obtain 30,000,000,000/gramBacillus subtilis wettable powder microbial inoculum.
Embodiment seven: the preparation three of bacillus subtilis wettable powder microbial inoculum
Following raw materials by weight is mixed: bacillus subtilis bacteria powder 25% that embodiment tetra-obtains,White carbon 10%, diatomite 10%, dodecyl sodium sulfate 10%, naphthalene sulfonate D-1002:15%, bentonite 20%, wettingAgent W-2001:10%. Then utilize more than airslide disintegrating mill is crushed to 400 orders, control moisture≤5%. Obtain 20,000,000,000/Gram bacillus subtilis wettable powder microbial inoculum.
Embodiment eight: the application of bacillus subtilis liquid bacterial agent control rice blast
8.1 reagent agent
Bacillus subtilis liquid bacterial agent prepared by the present invention
8.2 for studying thing and controlling object
For studying thing: paddy rice; Controlling object: rice blast
8.3 situations experimental field
Experimental field be located at Heilongjiang Province Songnen Plain, the Three River Plain and middle part rice terrace, soil types is slight natural pondPoolization meadow soil, the content of organic matter 2.1% left and right, pH6.2.
8.4 experimental design
This test taking 1,000,000,000/the bacillus subtilis liquid bacterial agent of milliliter designed stoste rare on this basis as stosteRelease 100 times, 500 times, 1000 times liquid and 4 processing of blank (clear water processing), every processing repeats 3 times, Gong12Ge community, withMachine district group is arranged, every community area 10 ㎡. 9.1～9.5 phases of paddy rice, boot stage and full heading time dispenser three times, do not exist together respectivelyReason all adopts cauline leaf spraying.
8.5 pilot surveies and computational methods
8.5.1 efficacy survey rice blast adopts the method investigation of diagonal sampling, and 5 points are got in every processing, get 1 at everyCave, repeats, calculates disease index and prevention effect for 3 times.
8.5.2 drug effect computational methods
8.6, result of the test
Reagent agent is as shown in table 4 to rice blast prevention effect, the bacillus subtilis liquid bacterial agent of 1,000,000,000/milliliterRice blast is had to obvious prevention effect, and wherein 100 times of liquid of the bacillus subtilis liquid bacterial agent of 1,000,000,000/milliliter dilution are anti-Control effect best, preventive effect reaches 64.7%.
The bacillus subtilis liquid bacterial agent control rice blast field effect test result of table 410 hundred million/milliliter
| Processed group | Average disease index (%) | Average prevention effect (%) |
| Clear water contrast | 40.03 | - |
| 100 times of liquid bacterial agent dilutions | 14.15 | 64.7 |
| 500 times of liquid bacterial agent dilutions | 16.23 | 59.5 |
| 1000 times of liquid bacterial agent dilutions | 17.86 | 55.4 |
8.7 brief summary
From rice blast disease exponential sum prevention effect, the bacillus subtilis liquid bacterial agent pair of 1,000,000,000/milliliterRice blast has good prevention effect, dilutes 100 times of liquid spray effect the bests, and prevention effect can reach 64.7%, diseaseEvil index is significantly less than control treatment.
Embodiment nine: the application of bacillus subtilis JQ-001 pulvis microbial inoculum control rice blast
9.1 reagent agent
Bacillus subtilis bacteria powder microbial inoculum prepared by the present invention
9.2 for studying thing and controlling object
For studying thing: paddy rice; Controlling object: rice blast
9.3 situations experimental field
Experimental field be located at Heilongjiang Province Songnen Plain, the Three River Plain and middle part rice terrace, soil types is slight natural pondPoolization meadow soil, the content of organic matter 2.1% left and right, pH6.2.
9.4 experimental design and arrangement
This experimental design 10,000,000,000/50 grams/acre, 150 grams/acre, 250 grams/acre of gram bacillus subtilis bacteria powder microbial inoculums and sky4 processing of white contrast, every processing repeats 3 times, Gong12Ge community, random district group is arranged, every community area 10 ㎡. Respectively in paddy rice9.1～9.5 phases, boot stage and full heading time dispenser three times, different disposal all adopts cauline leaf spraying.
9.5 pilot surveies and computational methods
9.5.1 efficacy survey rice blast adopts the method investigation of diagonal sampling, and 5 points are got in every processing, get 1 at everyCave, repeats, calculates disease index and prevention effect for 3 times.
9.5.2 drug effect computational methods
9.6 result of the test
Reagent agent is as shown in table 5 to rice blast prevention effect, 10,000,000,000/gram bacillus subtilis bacteria powder microbial inoculum pairRice blast has obvious prevention effect, wherein 250 grams/acre of prevention effects of 10,000,000,000/gram bacillus subtilis wettable powderBest, preventive effect reaches 78.2%.
Table 5,100 hundred million/gram pulvis microbial inoculum control rice blast field effect test result
| Processed group | Average disease index (%) | Average prevention effect (%) |
| Clear water contrast | 40.03 | - |
| 50 grams/acre of bacillus subtilis bacteria powders | 14.32 | 64.2 |
| 150 grams/acre of bacillus subtilis bacteria powders | 11.28 | 71.8 |
| 250 grams/acre of bacillus subtilis bacteria powders | 8.73 | 78.2 |
9.7 brief summary
From rice blast disease exponential sum prevention effect, 10,000,000,000/gram bacillus subtilis bacteria powder microbial inoculum is to paddy riceRice blast has good prevention effect, uses 250 grams of best results for every mu, and prevention effect can reach 78.2%, disease indexBe significantly less than control treatment.
Embodiment ten: the application of bacillus subtilis wettable powder microbial inoculum control rice blast
10.1 reagent agents
Bacillus subtilis wettable powder prepared by the present invention
10.2 for studying thing and controlling object
For studying thing: paddy rice; Controlling object: rice blast
10.3 experimental field situation
Experimental field be located at Heilongjiang Province Songnen Plain, the Three River Plain and middle part rice terrace, soil types is slight natural pondPoolization meadow soil, the content of organic matter 2.1% left and right, pH6.2.
10.4 experimental design and arrangement
This experimental design 20 grams/acre, 50 grams/acre, 80 grams/acre of 30,000,000,000/gram bacillus subtilis wettable powders and sky4 processing of white contrast, every processing repeats 3 times, Gong12Ge community, random district group is arranged, every community area 20 ㎡. Respectively in paddy rice9.1～9.5 phases, boot stage and full heading time dispenser three times. Different disposal all adopts cauline leaf spraying, 15 liters/mu of spouting liquids.
10.5 pilot surveies and computational methods
10.5.1 efficacy survey rice blast adopts the method investigation of diagonal sampling, and 5 points are got in every processing, get 1 at everyCave, repeats, calculates disease index and prevention effect for 3 times.
10.5.2 drug effect computational methods
10.6, result of the test
Reagent agent is as shown in table 6 to rice blast prevention effect, 30,000,000,000/gram bacillus subtilis wettable powderRice blast is had to obvious prevention effect, wherein 80 grams/acre of control effects of 30,000,000,000/gram bacillus subtilis wettable powderFruit is best, and preventive effect reaches 75.6%.
Table 6,300 hundred million/gram wettable powder control rice blast field effect test result
| Processed group | Average disease index (%) | Average prevention effect (%) |
| Clear water contrast | 40.03 | - |
| 20 grams/acre of wettable powders | 15.27 | 61.85 |
| 50 grams/acre of wettable powders | 10.69 | 73.30 |
| 80 grams/acre of wettable powders | 6.78 | 82.56 |
10.7 brief summaries
From rice blast disease exponential sum prevention effect, 30,000,000,000/gram bacillus subtilis wettable powder is to waterRice rice blast has good prevention effect, uses 80 grams of best results for every mu, and prevention effect can reach 82.56%, and disease refers toNumber is significantly less than control treatment.
Embodiment 11: JQ-001 bacterial strain is measured potato scab pathogen antagonistic effect
Solid medium: NA culture medium, Gause I culture medium: soluble starch 20g, KNO31g、NaCl0.5g、K2HPO40.5g、MgSO40.5g、FeSO40.01g agar powder 16g, distilled water 1000ml, pH7.4, when preparation, first use a small amount ofCold water, by starch furnishing pasty state, pours in the water boiling, and on fire, heats, and adds while stirring other composition, after thawing, mendsFoot moisture, to 1000ml, is then used 13 bottles of the average packing of 100ml conical flask, 121 DEG C of sterilizing 30min.
Fluid nutrient medium (withered grass fermentation medium): glucose 20g, Dried Corn Steep Liquor Powder 5g, peptone 1g, dusty yeast0.5g、MnSO420ml、MgSO410ml, running water 500ml, pH7.5,121 DEG C of sterilizing 30min.
One, experimental technique: adopt dull and stereotyped face-off cultivation
1, potato scab pathogenicbacteria separation purifying is cultivated
Sampling spot: in land for growing field crops, large whole village, Zhangbei County, Hebei province.
The strong intersection of disease of getting morbidity potato tubers with blade, first carries out after sterilizing processing with mercuric chloride and alcohol,In access Gause I culture medium, 4 of accesses in each culture dish, use card punch after cultivating 6 days in 25 DEG C of constant incubatorsGet long germ plate and carry out purifying cultivation, for subsequent use.
2, the preparation of bacillus subtilis JQ-001 zymotic fluid
Line on the bacterial classification oese access nutrient agar panel being kept on inclined-plane is cultivated to 37 DEG C of constant temperature culture24h activation. The bacterial strain access of activation is equipped with in the 500ml triangular flask of 50ml fermentation medium, 34 DEG C, 230r/min conditionUnder, shaking table shaken cultivation 25h, prepares strain fermentating liquid, produces completely after gemma until it, measure zymotic fluid number of viable and be 10,000,000,000/Milliliter.
3, fermentation liquor treatment
The zymotic fluid of having measured number of viable is diluted to 0.01,0.05,0.1,0.5,1.0,1.5,2.0 hundred million/milliliter bacteriumAmount, gets respectively each concentration bacterium liquid 3ml and sterilizing and is cooled to the Gause I culture medium of 45 DEG C of molten states with 1ml liquid-transfering gun(100ml conical flask quantitative separating 75ml for this culture medium) mixes in 1:25 ratio, makes the flat board that carries disease germs, and average every plate contains1ml bacterium liquid; Mix the flat board changing into for contrasting with the zymotic fluid of not inoculating bacillus subtilis JQ-001 with culture medium. At flat boardThe potato scab germ that middle access is cultivated in advance, gets bacterium piece with the card punch of diameter 5mm, accesses the culture dish solidifyingIn, every flat board is evenly inoculated two, and every processing repeats 3 times. In 25 DEG C of insulating boxs, back-off is cultivated 6 days.
4, experimental data processing
In the time contrasting mycelia and cover with plate, Taking Pictures recording, observes thalline growing way situation and also adopts right-angled intersection method to measure bacterium colonyDiameter, antibacterial circle diameter, calculating mean value and relative bacteriostasis rate, record colony diameter size, and more different bacterium amounts are to potatoThe inhibition of shot hole, calculates bacteriostasis rate according to the following formula.
Bacteriostasis rate (%)=(contrast colony diameter-processing colony diameter)/contrast colony diameter × 100%
5, result is shown
Table 7 bacillus subtilis JQ-001 is to potato scab pathogen antagonistic experiment result
Embodiment 12: JQ-001 bacterial strain bacterium liquid and pulvis are prevented and treated potato scab field experiment
This experimental design zymocyte liquid (viable bacteria content 10,000,000,000/milliliter) or use Dilution for powder become viable bacteria content 100The bacterium liquid of hundred million/milliliter directly sprays the plot experiment in soil, 5,000,000,000/square metre (processed group one), 2,000,000,000/square metre (processingGroup two), 1,000,000,000/square metre (processed group three) and blank (clear water processing) totally 4 processing, repeat 4 times, Gong16Ge community,Every community area 10m2, each community random alignment. In potato scab Seedling Stage, bloom squaring period, growth later stage pouring.
Biological and ecological methods to prevent plant disease, pests, and erosion rate=(the control group incidence of disease-test group incidence of disease)/control group incidence of disease × 100%
Incidence when table 8 bacillus subtilis JQ-001 bacterium liquid is processed potato
Prevention effect when table 9 bacillus subtilis JQ-001 bacterium liquid is processed potato
| Processed group | Average disease index (%) | Average prevention effect (%) |
| First group | 6.13 | 89.59 |
| Second group | 18.59 | 68.45 |
| The 3rd group | 21.52 | 63.48 |
As seen from the experiment, though in the case of zymocyte liquid or be equal to the bacterium liquid use amount of concentration on the low side stillShot hole is had to good test effect, and wherein the experiment effect of 5,000,000,000/square metre processing is best, has reached 89.59%.
Embodiment 13: JQ-001 bacterial strain wettable powder is prevented and treated potato scab field experiment
This experimental design 30,000,000,000/gram JQ-001 bacterial strain wettable powder 50 grams/acre (processed group four), 100 grams/acre (locateReason group five), 150 grams/acre (processed group six) and blank (clear water processing) totally 4 processing, repeat 4 times, Gong16Ge community, everyCommunity area 10m2, each community random alignment. Spray in potato scab premorbid, every dispenser in 7 days 1 time, dispenser 3 continuouslyInferior. Result is as follows:
Biological and ecological methods to prevent plant disease, pests, and erosion rate=(the control group incidence of disease-test group incidence of disease)/control group incidence of disease × 100%.
Incidence of disease result in table 10 wettable powder control experiment
| Processed group | Total potato piece number | Morbidity piece number | The incidence of disease |
| Clear water processing | 155 | 78 | 50.32％ |
| Processed group four | 157 | 54 | 34.39％ |
| Processed group five | 151 | 27 | 17.88％ |
| Processed group six | 149 | 11 | 7.38％ |
The average prevention effect of average disease exponential sum when table 11 wettable powder is processed
| Processed group | Average disease index (%) | Average prevention effect (%) |
| The 4th group | 34.39 | 31.66 |
| The 5th group | 17.88 | 64.47 |
| The 6th group | 7.38 | 85.33 |
Result shows, bacillus subtilis JQ-001 wettable powder provided by the invention can significantly reduce potato soreThe incidence of disease of scall, reach more than 85% the biological and ecological methods to prevent plant disease, pests, and erosion rate of potato scab, finally played and significantly improved potatoThe time of quality and its storage of guarantee.
Claims (9)
1. a bacillus subtilis, is characterized in that: bacillus subtilis JQ-001, is preserved in Chinese microorganism strain preservationAdministration committee's common micro-organisms center, depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation time: 2015In on November 9, in, preservation registration number is CGMCCNo.11632.
2. a bacillus subtilis microbial agent, is characterized in that: with bacillus subtilis JQ-001 claimed in claim 1 through plantThe microbial inoculum of making after sub-activation, seed culture, fermented and cultured.
3. bacillus subtilis microbial agent as claimed in claim 2, is characterized in that: described microbial inoculum comprises liquid bacterial agent and solidMicrobial inoculum.
4. a preparation method for bacillus subtilis microbial agent claimed in claim 2, specifically comprises:
A. actication of culture: the bacillus subtilis JQ-001 inoculation that low temperature is preserved is on solid medium, in 35-38 DEG CCultivate 34-38h, picking list bacterium colony is transferred on slant medium, in 35-38 DEG C of cultivation 16-24h, with cultivating under aseptic washingPrimary surface thalline is as inoculation liquid;
B. primary seed solution preparation: in seed culture medium, access inoculation liquid prepared by step a according to the inoculum concentration of 5%-10%,Rotating speed 200-260rpm, temperature 35-38 DEG C, shaking table is cultivated 16-24h, as primary seed solution;
C. secondary seed solution preparation: access one-level kind prepared by step b according to the inoculum concentration of 0.5%-3% in seed culture mediumSub-liquid, throughput is 0.4-1.5vvm, and mixing speed is 180-260rpm, and incubation time is 18-24h, as secondary seed solution;
D. fermented and cultured: in fermentation medium, access secondary seed solution prepared by step c according to the inoculum concentration of 5%-10%, logicalTolerance is 0.4-1.5vvm, and mixing speed is 180rpm-260rpm, and incubation time is 24h-30h, obtains zymotic fluid;
E. above-mentioned zymotic fluid is concentrated by washing and molecular sieve, obtain bacillus subtilis liquid bacterial agent.
5. the preparation method of bacillus subtilis microbial agent as claimed in claim 4, is characterized in that: described step b and step cIn constituent and the percentage by weight thereof of seed culture medium be: glucose 1-2%, peptone 1-1.5%, dusty yeast 0.6-1%, beef extract 0.5-0.7%, sodium chloride 0.5%, all the other are water.
6. the preparation method of bacillus subtilis microbial agent as claimed in claim 4, is characterized in that: sending out in described steps dThe constituent of ferment culture medium and percentage by weight thereof are: glucose 3-5%, peptone 0.5-1.5%, dusty yeast 0.03-0.08%, peptone 0.1-0.3%, bean cake powder 0.5-1.5%, magnesium sulfate 0.01-0.05%, manganese sulfate 0.01-0.05%, itsYu Weishui.
7. the preparation method of bacillus subtilis microbial agent as claimed in claim 4, is characterized in that: ferment described in steps dLiquid is sprayed dry, inlet temperature 150-200 DEG C, and outlet temperature 50-90 DEG C, adds spraying carrier soluble starch, obtainsBacillus subtilis bacteria powder.
8. the preparation method of bacillus subtilis microbial agent as claimed in claim 7, is characterized in that: according to percentage by weight,By raw material: bacillus subtilis bacteria powder 20-30%, white carbon 10-15%, diatomite 10-20%, dodecyl sodium sulfate 1-10%, naphthalene sulfonate D-10022-10%, bentonite 10-20% and wetting agent W-20012-10% mix, then powderMore than being broken to 400 orders, obtain bacillus subtilis wettable powder.
9. the application of bacillus subtilis microbial agent as claimed in claim 2, is characterized in that: described microbial inoculum is prevented harnessing the river in preparationApplication in rice rice blast and potato scab medicine.
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