CN105670973A - Bacillus methylotrophicus, bactericide and preparation method and application of bactericide - Google Patents
Bacillus methylotrophicus, bactericide and preparation method and application of bactericide Download PDFInfo
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Abstract
The invention relates to bacillus methylotrophicus, bactericide and a preparation method and application of the bactericide.The bacillus methylotrophicus is bacillus methylotrophicus JQ-018 and is preserved in China General Microbiological Culture Collection Center on 7th December, 2015, and the preservation register number is CGMCC No:11838.The bactericide can be prepared through seed activation, seed culture and fermentation culture.The invention further provides the preparation method of the bactericide of the bacillus methylotrophicus and the application to preparation of an agent for preventing and controlling potato early blight.The bactericide has high bioactivity and good environment compatibility, is free of poisons to people and livestock and convenient to use, and can adjust plant microorganism ecological florae.
Description
Technical field
The present invention relates to microbial technology field, be specifically related to the application of a kind of Methylotrophic bacillus cereus, microbial inoculum, bacterial preparation process and microbial inoculum.
Background technology
Target is a kind of disease that Rhizoma Solani tuber osi hazard ratio is more serious, and there is generation in the area of all plantation Rhizoma Solani tuber osis, has a strong impact on the yield of Rhizoma Solani tuber osi. Target is mainly caused by Alternaria alternaria solani sorauer, occurs mainly on blade, it is possible to infect tuber. The spore that on leaf of potato, scab produces, by dispersive propagation such as wind, rain, insecticides, can infect the healthy plant of surrounding. When blade face moistens, drop to the spore germination on blade, pore and wound invade, be formed for new scab after a few days, scab produces again spore, dispersive propagation. In a season of growth, it is possible to repeatedly occur repeatedly to infect, so that causing full field to fall ill. Rainfall is also beneficial to Sporulation, after rain 2～3 days, the spore quantity showed increased dispersed in the air, behind spore dispersal peak period 10～20 days, and field morbidity quantity sharply increases. Serious blade of falling ill is withered to come off, and field is a piece of withered and yellow, causes serious economic loss.
At present this disease is prevented and treated mainly by spraying antibacterial at the initial stage of a disease in time. Institute's with medicament mainly has 600 times of liquid of 75% Bravo wettable powder or 500 times of liquid of 64% Sandofan wettable powder, 80% spray 800 times of liquid of gram wettable powder, 80% raw greatly 600 times of liquid of M-45 wettable powder, 500 times of liquid of 70% mancozeb wettable powder, 600 times of liquid of 80% Xin Wansheng wettable powder, 1:1:200 times of formula Bordeaux mixture, 77% can kill to obtain 500 times of liquid of wettability microgranule powder. Life-time service chemical bactericide, causes that pathogenic bacteria Drug resistance strengthens, and only strengthens dosage and shortens spray date interval and control disease, and not only increasing production cost, to also lead to persticide residue more and more much. Potato tubers expands needs loose fertile soil, uses chemical agent in a large number, it is easy to cause soil pollution, the acid-base disorder of soil, and nutrient content reduces, and soil compaction has a strong impact on the yield of crop.Therefore Biological control is adopted to control the inexorable trend that target is agricultural development. And adopt antagonistic microbe controlling disease to be that instead of the one new method safe and nontoxic, effective of chemical agent. Antagonistic microbe is possible not only to suppress the growth of pathogen, moreover it is possible to increase the quantity of Soil Microorganism, promotes plant growing, to improving ecosystem, keeps bio-diversity, and maintaining ecological balance has important effect.
By literature search, there is not yet and utilize Methylotrophic bacillus cereus prepare bacteria agent and be applied to the report of control in field target.
Summary of the invention
It is an object of the invention to provide a kind of new Methylotrophic bacillus cereus.
Another object of the present invention is to provide a kind of with Methylotrophic bacillus cereus be active component microbial inoculum and preparation method.
Further object of the present invention be in that provide utilize with Methylotrophic bacillus cereus be active component microbial inoculum preparation controlling plant diseases medicine in application.
It is an object of the invention to be achieved through the following technical solutions:
A kind of Methylotrophic bacillus cereus, it is characterized in that: Methylotrophic bacillus cereus (Bacillusmethylotrophicus) JQ-018, it 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, the preservation time: on December 7th, 2015, preservation registration number is CGMCCNo.11838.
The Methylotrophic bacillus cereus JQ-018 of the present invention separates from big whole village, Zhangbei County, Hebei province Rhizoma Solani tuber osi foundation soil, purification and screening step obtain, and adopting morphological characteristic, bio-chemical characteristics, molecular biology identification method and 16SrDNA homologous sequence comparison to analyze the bacterial strain finally determining above-mentioned deposit number is Methylotrophic bacillus cereus (Bacillusmethylotrophicus).
The microbial inoculum being active component with Methylotrophic bacillus cereus, is strain by Methylotrophic bacillus cereus JQ-018, through the microbial inoculum that seed activation, seed culture, fermentation culture obtain; Including liquid bacterial agent and solid fungicide.
With the preparation method of the microbial inoculum that Methylotrophic bacillus cereus is active component, specifically comprise the following steps that
A actication of culture: by the Methylotrophic bacillus cereus JQ-018 inoculation of cryopreservation on solid medium, cultivates 34-38h in 35-38 DEG C; Picking list bacterium colony is transferred on slant medium, cultivates 16-24h in 35-38 DEG C, with media surface thalline under aseptic washing as inoculation liquid;
Prepared by b primary seed solution: accessing the step a inoculation liquid prepared, rotating speed 200-260rpm according to the inoculum concentration of 5-10% in seed culture medium, temperature 35-38 DEG C, 16-24h cultivated by shaking table, as primary seed solution;
Prepared by c secondary seed solution: accessing the step b primary seed solution prepared according to the inoculum concentration of 0.5-3% in seed culture medium, ventilation is 0.4-1.5vvm, and mixing speed is 180-260rpm, and incubation time is 18-24h, as secondary seed solution;
The constituent of described seed culture medium and percentage by weight thereof be: glucose 1-2%, peptone 1-1.5%, yeast powder 0.6-1%, Carnis Bovis seu Bubali cream 0.5-0.7%, sodium chloride 0.5%, and all the other are water;
D fermentation culture: accessing the step c secondary seed solution prepared according to the inoculum concentration of 5-10% in fermentation medium, ventilation is 0.4-1.5vvm, and mixing speed is 180-260rpm, and incubation time is 24-30h, obtains fermentation liquid;
The constituent of described fermentation medium and percentage by weight thereof be: glucose 3-5%, peptone 0.5-1.5%, yeast powder 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%, and all the other are water;
Above-mentioned fermentation liquid is carried out 80-160 order filtration by e, obtains Methylotrophic bacillus cereus liquid microbial inoculum.
F is by above-mentioned fermented liquid spray drying: inlet temperature 150-200 DEG C, outlet temperature 50-90 DEG C, adds spray carrier soluble starch. Obtain Methylotrophic bacillus cereus powder.
G is by following raw materials by weight mix homogeneously: Methylotrophic bacillus cereus powder 10-30%, white carbon 10-30%, kieselguhr: 10-30%, dodecyl sodium sulfate 1-10%, naphthalene sulfonate D-1002:2-10%, bentonite 10-30%, wetting agent W-2001:2-10%. Then utilize jet mill to be crushed to more than 400 orders, control moisture≤5%. Obtain Methylotrophic bacillus wettable powder microbial inoculum.
Further, the present invention also provides for the application that three of the above Methylotrophic gemma bacillus agent is prevented and treated in target medicine in preparation.
The method have the advantages that
Methylotrophic bacillus cereus of the present invention is safe and nontoxic, the liquid bacterial agent prepared by it has the advantages that the fermenting and producing cycle is short, cost is low, it is beneficial to industrialized production, suitable in crop root irrigation, it is constantly in liquid condition, so the activity of bacterium is maintained at higher state when producing because of it and use; Make powder, the preservation cycle can be extended after particularly preparing into wettable powder, after sprinkling microbial inoculum can field planting in crop surface, capture the pathogen Ecological niche, it is suppressed that growth of pathogenic bacteria, pathogen suppression ratio is reached more than 85%, convenient use, and plant growth can be promoted, increase crop yield, maintaining ecological balance.
Culture presevation
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center; Unit address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; The preservation time: on December 7th, 2015; Preservation registration number is CGMCCNo.11838.
Accompanying drawing explanation
Dendrogram is analyzed in the 16SrDNA homologous sequence comparison that Fig. 1 is the Methylotrophic bacillus cereus of the present invention.
Detailed description of the invention
Further illustrate below in conjunction with being embodied as row:
Embodiment one: the separation of Methylotrophic bacillus cereus JQ-018 bacterial strain and screening:
1, separate
(1) soil collecting: gather soil sample 100g, 5 samples of the three unities parallel acquisition from upper soll layer in big whole village, Zhangbei County, Hebei province potato fields to 10cm, mixed 40 order classifying screens. Take the lower soil of 100g sieve and be used as separation.
(2) culture medium preparation: prepare respectively
NA culture medium: peptone 10g/L, Carnis Bovis seu Bubali cream 3.0g/L, sodium chloride 5g/L, agar 18g/LpH=7.0-7.2;
With PDA culture medium: Rhizoma Solani tuber osi 200g, glucose 20g, agar powder 20g, distilled water 1000mL;
Make culture medium flat plate.
(3) accurately weigh 1g soil and be placed in 250ml triangular flask, add 99ml sterilized water, shaking table 200rpm, 37 DEG C of concussion 30min. Make Soil Slurry, by 10 times of gradient dilutions to 10-8. Separating single bacterium colony respectively in NA culture medium and PDA culture medium, the single bacterium colony being separated to activates 4 cycles respectively in NA culture medium and PDA culture medium.
2, screening
The single bacterium colony and other strain list bacterium colony that separate activation are carried out flat board face-off experiment with target pathogen, the following is screening process face-off experiment and result thereof:
The pathogen (target) cultivated in advance is accessed in PDA culture medium flat plate central authorities, bacterial plaque is got with the card punch of diameter 9mm during long size to fit to be generated, move to new PDA culture medium flat plate central authorities, at distance pathogen bacterial plaque 2cm place inoculation strain, each process is repeated 3 times, it is placed in 25 DEG C of biochemical cultivation cases to cultivate, observes each colony diameter, measure bacteriostasis rate.
Bacteriostasis rate (%)=(comparison colony diameter-process colony diameter)/comparison colony diameter × 100%
Table 1JQ-018 and other bacterial strain colony diameter (cm)
| Strain name | Parallel processing 1 | Parallel processing 2 | Parallel processing 3 | On average |
| JQ-011 | 0.5 | 0.5 | 0.6 | 0.53 |
| JQ-012 | 0.0 | 0.0 | 0.0 | 0.0 |
| JQ-013 | 0.4 | 0.3 | 0.2 | 0.3 |
| JQ-014 | 0.6 | 0.6 | 0.6 | 0.6 |
| JQ-015 | 0.7 | 0.8 | 0.6 | 0.7 |
| JQ-016 | 0.0 | 0.0 | 0.0 | 0.0 |
| JQ-017 | 0.3 | 0.3 | 0.3 | 0.3 |
| JQ-018 | 0.9 | 0.9 | 1.0 | 0.93 3 --> |
| JQ-019 | 0.7 | 0.7 | 0.6 | 0.6 |
| JQ-020 | 0.1 | 0.2 | 0.1 | 0.13 |
JQ-018 bacterial strain is the highest to the bacteriostatic activity of target as can be seen from Table 1. The antagonistic experiment continuing to do JQ-018 bacterial strain obtains following result, in Table 2.
Table 2JQ-018 bacterial strain is to target Indoor antibacterial determination of activity
Embodiment two: the qualification of Methylotrophic bacillus cereus JQ-018 bacterial strain:
1, Microbiological Characteristics: JQ-018 thalline is rod-short, peritrichous, can produce spore, Gram-positive. Bacterial strain is in PDA culture medium after 34 DEG C of cultivation 12h, and bacterium colony is circular, translucent white, smooth surface, intermediate projections, not chromogenesis, toughness. In NA culture medium, bacterium colony is less or do not grow. Liquid culture forms Mycoderma, produces spore, brown color, muddiness, have precipitation after 24h. Strains expressed aerobic growth.
2, bio-chemical characteristics:
The physiological and biochemical property of table 3 Methylotrophic bacillus cereus JQ-018
| Detection project | Characteristic | Detection project | Characteristic | Detection project | Characteristic |
| D-Fructose | + | Salicin | - | Mannitol | + |
| Maltose | + | Polysorbate40 | - | L MALIC ACID | + |
| D-galactose | + | D-fructose-6-phosphoric acid | - | Acetone acid | + |
| D-Glucose | + | Rhamnose | - | Agedoite | + |
Note: "+" representing positive reaction, "-" represents negative reaction
3, molecular biology identification:
The base sequence of 1414bp length is obtained after the order-checking of JQ-018 bacterial strain, by the comparison in ncbi database of this sequence, 99.38% is reached with Bacillusmethylotrophicus homology, in conjunction with the morphological characteristic of JQ-018 bacterial strain, physio-biochemical characteristics, determining that the JQ-018 bacterial strain detected belongs to Methylotrophic bacillus cereus, the phylogenetic tree of the JQ-018 bacterial strain based on 16SrDNA sequence is (in figure, bacterial strain 3 is JQ-018 bacterial strain) as shown in Figure 1.
Embodiment three: the preparation of Methylotrophic bacillus cereus liquid microbial inoculum
(1) actication of culture: by the Methylotrophic bacillus cereus JQ-018 inoculation of cryopreservation on LB solid medium, cultivates 36h in 36 DEG C; Picking list bacterium colony is transferred on LB slant medium, cultivates 20h in 36 DEG C, with media surface thalline under aseptic washing as inoculation liquid.
(2) prepared by primary seed solution: accessing inoculation liquid prepared by step (1), rotating speed 250rpm, temperature 36 DEG C according to the inoculum concentration of 10% (percent by volume) in LB seed culture medium, 20h cultivated by shaking table, as primary seed solution.
(3) prepared by secondary seed solution: accessing primary seed solution prepared by step (2) according to the inoculum concentration of 3% (percent by volume) in seed culture medium, ventilation is 1.0vvm, and mixing speed is 220rpm, and incubation time is 20h. As secondary seed solution.
The constituent of described seed culture medium and percentage by weight thereof be: glucose 2%, peptone 1.5%, yeast powder 0.5%, Carnis Bovis seu Bubali cream 0.6%, sodium chloride 0.5%, all the other are water.
(4) fermentation culture: access secondary seed solution prepared by step (3) in fermentation medium according to the inoculum concentration of 10% (percent by volume), ventilation is 1.0vvm, mixing speed is 220rpm, and incubation time is 28h, obtains fermentation liquid.
The constituent of described fermentation medium and percentage by weight thereof be: glucose 5%, peptone 1.5%, yeast powder 0.05%, peptone 0.2%, bean cake powder 1.0%, magnesium sulfate 0.03%, manganese sulfate 0.03%, all the other are water.
(5) above-mentioned fermentation liquid is carried out 100 order filtrations, obtain the Methylotrophic bacillus cereus liquid microbial inoculum that viable count is more than 100,000,000,000/milliliter.
Embodiment four: the preparation of Methylotrophic bacillus cereus powder microbial inoculum
Fermentation liquid obtained for embodiment three is carried out spray drying: inlet temperature 180 DEG C, outlet temperature 80 DEG C, add spray carrier soluble starch. Gained microbial inoculum living bacteria count is 100,000,000,000/gram more than.
Embodiment five: the preparation one of Methylotrophic bacillus wettable powder microbial inoculum
By following raw materials by weight mix homogeneously: the obtained Methylotrophic bacillus cereus powder 30% of embodiment four, white carbon 30%, kieselguhr 10%, dodecyl sodium sulfate 10%, naphthalene sulfonate D-1002:2%, bentonite 10%, wetting agent W-2001:8%. Then utilize jet mill to be crushed to more than 400 orders, control moisture≤5%. Obtain 40,000,000,000/gram Methylotrophic bacillus wettable powder microbial inoculum.
Embodiment six: the preparation two of Methylotrophic bacillus wettable powder microbial inoculum
By following raw materials by weight mix homogeneously: the obtained Methylotrophic bacillus cereus powder 20% of embodiment four, white carbon 29%, kieselguhr 10%, dodecyl sodium sulfate 1%, naphthalene sulfonate D-1002:10%, bentonite 20%, wetting agent W-2001:10%. Then utilize jet mill to be crushed to more than 400 orders, control moisture≤5%. Obtain 30,000,000,000/gram Methylotrophic bacillus wettable powder microbial inoculum.
Embodiment seven: the preparation three of Methylotrophic bacillus wettable powder microbial inoculum
By following raw materials by weight mix homogeneously: the obtained Methylotrophic bacillus cereus powder 10% of embodiment four, white carbon 10%, kieselguhr: 30%, dodecyl sodium sulfate 10%, naphthalene sulfonate D-1002:8%, bentonite 30%, wetting agent W-2001:2%. Then utilize jet mill to be crushed to more than 400 orders, control moisture≤5%. Obtain 20,000,000,000/gram Methylotrophic bacillus wettable powder microbial inoculum.
Embodiment eight: the application of Methylotrophic bacillus cereus liquid microbial inoculum
8.1 reagent agents
Methylotrophic bacillus cereus liquid microbial inoculum prepared by the present invention
8.2 for studying thing and controlling object
It is Rhizoma Solani tuber osi for studying thing, controlling object: target 8.3 experimental field situation
Experimental field being located at big whole village, Zhangbei County, Hebei province, height above sea level 1600-1800 rice, soil is sand, organic 1.15%-1.35%.
8.4 EXPERIMENTAL DESIGN and arrangement
This test is with 1,000,000,000/milliliter Methylotrophic bacillus cereus liquid microbial inoculum for stock solution, and 100 times, 500 times, 1000 times liquid of dilution carry out root irrigation on this basis, processes as comparison using clear water, set 4 process altogether, repeat 4 times, Gong16Ge community, every plot area 5m2, each community random alignment. In target premorbid pouring root, processed 1 time every 7 days, process 3 times continuously.
8.5 test surveys and computational methods
Last processes latter 7 days investigation disease indexs and calculates preventive effect. Each community randomly selects 5 samplings, and every some investigation 3 strains, whole blades are investigated in every strain, and the percentage rate accounting for whole leaf area with the lesion area on every leaf carrys out classification. Disease scale standard is as follows: 0 grade: without scab; 1 grade: single blade has scab 3; 3 grades: single blade has scab 4-6; 5 grades: single blade has scab 7-10;7 grades: single blade has scab more than 10, and part is intensive in flakes; 9 grades: single leaf spot lesion is intensive, and yellow leaf is withered.
8.6 results
Target field test results prevented and treated by table 41,000,000,000/milliliter Methylotrophic bacillus cereus liquid microbial inoculum
| Process group | Mean disease index (%) | Average prevention effect (%) |
| Clear water compares | 40.17 | - |
| Liquid bacterial agent dilutes 100 times | 13.12 | 67.3 |
| Liquid bacterial agent dilutes 500 times | 11.83 | 70.6 |
| Liquid bacterial agent dilutes 1000 times | 15.34 | 61.8 |
Table 4 result shows, after the different times liquid of 1000000000/milliliter Methylotrophic bacillus cereus liquid microbial inoculum dilution, target is had good prevention effect by root irrigation, wherein 1,000,000,000/milliliter Methylotrophic bacillus cereus liquid microbial inoculum dilutes 500 times of liquid prevention effects preferably, and prevention effect has reached 70.6%.
8.7 brief summaries
From Rhizoma Solani tuber osi disease index and prevention effect, after the different times liquid of 1000000000/milliliter Methylotrophic bacillus cereus liquid microbial inoculum dilution, target is had good prevention effect by root irrigation, dilute 500 times of liquid treatment effects best, prevention effect can reach 70.6%, and disease index is significantly less than control treatment.
Embodiment nine: the application of Methylotrophic bacillus cereus powder microbial inoculum
9.1 reagent agents
Methylotrophic bacillus cereus powder microbial inoculum prepared by the present invention
9.2 for studying thing and controlling object
It is Rhizoma Solani tuber osi for studying thing, controlling object: target 9.3 experimental field situation
Experimental field being located at big whole village, Zhangbei County, Hebei province, height above sea level 1600-1800 rice, soil is sand, organic 1.15%-1.35%.
9.4 EXPERIMENTAL DESIGN and arrangement
This EXPERIMENTAL DESIGN 30,000,000,000/gram Methylotrophic bacillus cereus powder microbial inoculum 50g dresses seed potato seed pieces 0.5kg, 1kg, 2kg and blank 4 process totally, repeats 4 times, Gong16Ge community, every plot area 5m2, each community random alignment. The long-term Routine Management of potato raw.
9.5 test surveys and computational methods
After planting add up and manage germination percentage everywhere. Period of disease investigation disease index also calculates preventive effect, and each community randomly selects 5 samplings, and every some investigation 3 strains, whole blades are investigated in every strain, and the percentage rate accounting for whole leaf area with the lesion area on every leaf carrys out classification. Disease scale standard is as follows: 0 grade: without scab; 1 grade: single blade has scab 3; 3 grades: single blade has scab 4-6; 5 grades: single blade has scab 7-10; 7 grades: single blade has scab more than 10, and part is intensive in flakes; 9 grades: single leaf spot lesion is intensive, and yellow leaf is withered.
9.6 results
A table 530,000,000,000/gram Methylotrophic bacillus cereus powder microbial inoculum seed dressing prevents and treats target field test results
| Process group | Germination percentage (%) | Mean disease index (%) | Average prevention effect (%) |
| Blank | 93 | 43.75 | - |
| Powder microbial inoculum 50g dresses seed 0.5kg | 95 | 20.68 | 52.7 |
| Bacteria powder microbial inoculum 50g dresses seed 1kg | 98 | 21.15 | 51.7 |
| Bacteria powder microbial inoculum 50g dresses seed 2kg | 96 | 24.73 | 43.5 |
Table 5 result shows, a 30000000000/gram Methylotrophic bacillus cereus powder microbial inoculum Dressing germination percentage is all higher than blank and processes, wherein 30,000,000,000/gram Methylotrophic bacillus cereus powder microbial inoculum 50g seed dressing 1kg potato seed pieces germination percentage is the highest, reaches 98%; And target is had good prevention effect by a 30000000000/gram Methylotrophic bacillus cereus powder microbial inoculum Dressing, wherein 30,000,000,000/gram Methylotrophic bacillus cereus powder microbial inoculum 50g dresses seed 0.5kg potato seed pieces prevention effect preferably, has reached 52.7%. To sum up, in conjunction with germination percentage and prevention effect two indices, better by 30,000,000,000/gram Methylotrophic bacillus cereus powder microbial inoculum 50g seed dressing 1kg potato seed pieces effect.
9.7 brief summaries
From potato sprouting rate, a 30000000000/gram Methylotrophic bacillus cereus powder microbial inoculum Dressing germination percentage is all higher than blank and processes, wherein 30,000,000,000/gram Methylotrophic bacillus cereus powder microbial inoculum 50g seed dressing 1kg potato seed pieces germination percentage is the highest, reaches 98%.
From Rhizoma Solani tuber osi disease index and prevention effect, target is had good prevention effect by a 30000000000/gram Methylotrophic bacillus cereus powder microbial inoculum Dressing, wherein 30,000,000,000/gram Methylotrophic bacillus cereus powder microbial inoculum 50g seed dressing 0.5kg potato seed pieces effect is best, prevention effect can reach 52.7%, and disease index is significantly less than control treatment.
To sum up, in conjunction with germination percentage and prevention effect two indices, better by 30,000,000,000/gram Methylotrophic bacillus cereus powder microbial inoculum 50g seed dressing 1kg potato seed pieces effect.
Embodiment ten: the application of Methylotrophic bacillus cereus JQ-018 wettability microbial inoculum
10.1 reagent agent
Methylotrophic bacillus cereus JQ-018 bacterial strain wettable powder prepared by the present invention
10.2 for studying thing and controlling object
It is Rhizoma Solani tuber osi for studying thing, controlling object: target
10.3 experimental field situation
Experimental field being located at big whole village, Zhangbei County, Hebei province, height above sea level 1600-1800 rice, soil is sand, organic 1.15%-1.35%.
10.4 EXPERIMENTAL DESIGN and arrangement
This EXPERIMENTAL DESIGN 20,000,000,000/gram Methylotrophic bacillus cereus (JQ-018 bacterial strain) wettable powder 50 grams/acre, 100 grams/acre, 150 grams/acre and blank (clear water process) totally 4 process, repeat 4 times, Gong16Ge community, every plot area 10m2, each community random alignment. Spray in target premorbid, every dispenser in 7 days 1 time, continuous dispenser 3 times.
10.5 test survey and computational methods
Within after last dispenser 7 days, investigate disease indexs and calculate preventive effect. Each community randomly selects 5 samplings, and every some investigation 3 strains, whole blades are investigated in every strain, and the percentage rate accounting for whole leaf area with the lesion area on every leaf carrys out classification. Disease scale standard is as follows: 0 grade: without scab; 1 grade: single blade has scab 3; 3 grades: single blade has scab 4-6; 5 grades: single blade has scab 7-10; 7 grades: single blade has scab more than 10, and part is intensive in flakes; 9 grades: single leaf spot lesion is intensive, and yellow leaf is withered.
10.6 result
A table 620,000,000,000/gram Methylotrophic bacillus wettable powder prevents and treats target field test results
| Process group | Mean disease index (%) | Average prevention effect (%) |
| Clear water compares | 39.43 | - |
| Wettable powder 50 grams/acre | 12.17 | 69.1 |
| Wettable powder 100 grams/acre | 8.62 | 78.1 |
| Wettable powder 150 grams/acre | 5.19 | 86.8 |
Table 6 result shows, target is had good prevention effect by 20000000000/gram Methylotrophic bacillus wettable powder, wherein 150 grams/acre of prevention effects of 20,000,000,000/gram Methylotrophic bacillus wettable powder are best, and prevention effect has reached 86.8%.
10.7 brief summary
From Rhizoma Solani tuber osi disease index and prevention effect, target is had good prevention effect by 20000000000/gram Methylotrophic bacillus wettable powder, using 150 grams of best results for every mu, prevention effect can reach 86.8%, and disease index is significantly less than control treatment.
Claims (9)
1. a Methylotrophic bacillus cereus, it is characterized in that: Methylotrophic bacillus cereus JQ-018, it 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, the preservation time: on December 7th, 2015, preservation registration number is CGMCCNo.11838.
2. the microbial inoculum that a kind is active component with Methylotrophic bacillus cereus, it is characterised in that: with the Methylotrophic bacillus cereus JQ-018 described in claim 1 for strain, through the microbial inoculum that seed activation, seed culture, fermentation culture obtain.
3. the microbial inoculum being active component with Methylotrophic bacillus cereus as claimed in claim 2, it is characterised in that: described microbial inoculum includes liquid bacterial agent and solid fungicide.
4. a preparation method for the microbial inoculum being active component with Methylotrophic bacillus cereus described in claim 2, specifically includes:
A. actication of culture: by the Methylotrophic bacillus cereus JQ-018 inoculation of cryopreservation on solid medium, cultivates 34-38h in 35-38 DEG C; Picking list bacterium colony is transferred on slant medium, cultivates 16-24h in 35-38 DEG C, with media surface thalline under aseptic washing as inoculation liquid;
B. prepared by primary seed solution: accessing the step a inoculation liquid prepared, rotating speed 200-260rpm according to the inoculum concentration of 5-10% in seed culture medium, temperature 35-38 DEG C, 16-24h cultivated by shaking table, as primary seed solution;
C. prepared by secondary seed solution: accessing the step b primary seed solution prepared according to the inoculum concentration of 0.5-3% in seed culture medium, ventilation is 0.4-1.5vvm, and mixing speed is 180-260rpm, and incubation time is 18-24h, as secondary seed solution;
D. fermentation culture: access the step c secondary seed solution prepared in fermentation medium according to the inoculum concentration of 5-10%, ventilation is 0.4-1.5vvm, mixing speed is 180-260rpm, and incubation time is 24-30h, obtains Methylotrophic bacillus cereus JQ-018 bacterial strain fermentation liquor;
E. above-mentioned fermentation liquid is filtered, obtains described Methylotrophic bacillus cereus liquid microbial inoculum.
5. the preparation method of the microbial inoculum being active component with Methylotrophic bacillus cereus as claimed in claim 4, it is characterized in that: the constituent of the seed culture medium in described step b and step c and percentage by weight thereof be: glucose 1-2%, peptone 1-1.5%, yeast powder 0.6-1%, Carnis Bovis seu Bubali cream 0.5-0.7%, sodium chloride 0.5%, all the other are water.
6. the preparation method of the microbial inoculum being active component with Methylotrophic bacillus cereus as claimed in claim 4, it is characterized in that: the constituent of the fermentation medium in described step d and percentage by weight thereof be: glucose 3-5%, peptone 0.5-1.5%, yeast powder 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.
7. the preparation method of the microbial inoculum being active component with Methylotrophic bacillus cereus as claimed in claim 4, it is characterized in that: fermentation liquid described in step d is carried out spray drying, inlet temperature 150-200 DEG C, outlet temperature 50-90 DEG C, add spray carrier soluble starch, obtain Methylotrophic bacillus cereus powder.
8. the preparation method of the microbial inoculum being active component with Methylotrophic bacillus cereus as claimed in claim 7, it is characterized in that: according to percentage by weight, by raw material: Methylotrophic bacillus cereus powder 10-30%, white carbon 10-30%, kieselguhr 10-30%, dodecyl sodium sulfate 1-10%, naphthalene sulfonate D-10022-10%, bentonite 10-30% and wetting agent W-20012-10% mix homogeneously, then it is crushed to more than 400 orders, obtains Methylotrophic bacillus wettable powder.
9. the application of the microbial inoculum being active component with Methylotrophic bacillus cereus as claimed in claim 2, it is characterised in that: described microbial inoculum prevents and treats the application in target medicine in preparation.
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