CN102154186B - Bacillus subtilis and use thereof in prevention and control of fungus disease - Google Patents
Bacillus subtilis and use thereof in prevention and control of fungus disease Download PDFInfo
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- CN102154186B CN102154186B CN 201110093415 CN201110093415A CN102154186B CN 102154186 B CN102154186 B CN 102154186B CN 201110093415 CN201110093415 CN 201110093415 CN 201110093415 A CN201110093415 A CN 201110093415A CN 102154186 B CN102154186 B CN 102154186B
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Abstract
The invention discloses Bacillus subtilis, which is named Bacillus subtilis. The Bacillus subtilis was collected in China General Microbiological Culture Collection Center on January 15th, 2009, with a collection number of CGMCC No.2843. The Bacillus subtilis strain disclosed by the invention is screened from tobacco leaf habitats. Experiments prove that the fermentation liquor of the strain has a strong inhibition effect on fungal pathogens such as Alternaria alternate and pepper anthracnose and demonstrates high prevention effect on Alternaria alternate, tobacco black shank, pepper anthracnose, tomato wilt, phytophthora melonis Katsura, verticillium dahlia and the like and has high growth promoting effects on tobacco, pepper, cucumber, tomato and eggplant. Therefore, the Bacillus subtilis disclosed by the invention can be used for preparing biological bactericides for preventing and controlling fungi in tobacco and vegetables and can be used for preparing growth promoting agents for promoting the growth of tobacco or vegetables.
Description
Technical field
The present invention relates to a bacillus subtilis, and the application in tobacco or vegetable fungi disease control, promotion tobacco or vegetable growth, the biological control field belonged to.
Background technology
For a long time, Plant diseases is main with chemical prevention, but receives that the bactericide agricultural chemicals kind of high-efficiency low-toxicity is few, preventive effect is subject to environmental influence, is prone to cause environmental pollution and problem puzzlement such as develop immunity to drugs.Biological control has the advantage to environment, ecology and human health safety, in integrated pest management, has brought into play increasing effect, and is considered to realize the important means of agricultural sustainable development.The concern that Along with people's is environmentally safe and healthy; Specification of quality to tobacco (sucking article) and vegetable prods is also increasingly high; Nuisanceless, green food even Organic food become the main flow of consumption market gradually, therefore, adopt the biological control technology to obtain approval widely.In recent years, extensively utilize useful microbe and meta-bolites thereof to suppress pathogenic bacteria existence and movable both at home and abroad, make this prophylactico-therapeutic measures be able to fast development.
The U.S. has 4 bacillus subtilis biocontrol strains such as GB03, MBI600, the mutation of QST713 reconciliation starch subtilis and obtains Environmental Protection Agency's commercialization or limited commercialization production application permission.Separate starch subtilis mutation FZB24 in Germany and commercially produced, be used to control the different fungal diseases of food crop.China has succeeded in developing and the subtilis commodity preparation that puts into production has inferior precious, hundred anti-, wheat Fengning, line Qu Ning etc.Said product is mainly used in the control of grain and fruit tree fungal disease, and Shang Weijian is used for the report of tobacco, capsicum, cucumber, eggplant, tomato fungal disease.
The domestic antagonistic bacterium of fungal diseases such as control Alternaria alternate, balck shank of having reported mainly contains Pseudomonas fluorescence (Gu Jingang etc., 2004; Li Honglin etc., 2008), Pseudomonas chlororaphis (Wang Yuanshan etc., 2002), bacillus pumilus AR03 (Wang Jing, 2010), subtilis (Yi Long etc., 2007; Zhang Cheng economizes and waits 2008), control capsicum epidemic disease antagonistic bacterium bacillus amyloliquefaciens (Liu Feng, 2009), vegetable fungi disease antagonistic bacteriums such as control cucumber fusarium axysporum, eggplant verticillium wilt, leaf muld of tomato do not appear in the newspapers.Above-mentioned research only is biocontrol strain screening and mechanism research, is not made into the preparation that is applicable to suitability for industrialized production and application, and there is a difficult problem in its practical application.
The inventor has made great efforts to have developed a kind ofly to have strong anti-mycotic activity, promote plant-growth, prevents and treats fungal disease, improves the multifunctional novel microbial bactericide of quality of tobacco, and said Plant diseases comprises Alternaria alternate, black shank, capsicum epidemic disease, leaf muld of tomato, eggplant verticillium wilt and cucumber fusarium axysporum.Through retrieval, in the patent documentation that all have been applied for, all do not relate to the bibliographical information of theme of the present invention.
Summary of the invention
To above-mentioned prior art, the invention provides a bacillus subtilis, it has preventive and therapeutic effect to tobacco or vegetable fungi disease, and the growths of tobacco or vegetables is had promoter action, and the raising of quality of tobacco is also had certain effect.The present invention also provides a kind of microbial preparation that is prepared from this spawn culture.
The present invention realizes through following technical scheme:
One bacillus subtilis, this bacterial strain called after: subtilis Bacillus subtilis, carry out preservation on December 29th, 2008 by China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCCNo.2843.
Subtilis Tpb55 involved in the present invention, its isolation identification, cultural method, biological nature, fermentation condition etc., open; Be documented in Chinese document " tobacco leaf encloses the evaluation and the bacteriostatic activity thereof of bacterium Tpb55 bacterial strain, Chinese biological control, 2008; 24 (1): 63-68 " and " screening of Alternaria alternate antagonistic bacterium Tpb55 conditions of flask fermentation; Chinese tobacco science 2009,30 (1): 54-57 ", repeat no more at this.
A kind of microbial preparation is made up of the full nutrient solution culture of subtilis Bacillus subtilis Tpb55 and the spore of subtilis Bacillus subtilis Tpb55; Said preparation has the disease prevention growth-promoting effect to tobacco or vegetable fungi disease.It can prepare through following preparation method:
(1) subtilis Bacillus subtilis Tpb55 test tube kind is inoculated in broth culture (be conventional substratum, its prescription repeats no more at this for known) and shakes in the bottle shaking culture to logarithmic phase;
(2) with the volume ratio 10% inoculum size access 30L seeding tank of above-mentioned cultured bacterial classification by the seed tank culture base, be cultured to logarithmic phase, the used culture medium prescription of seeding tank is: glucose 10g/L, peptone 3g/L, MgSO
40.5g/L, yeast extract paste 1.5g/L, Carnis Bovis seu Bubali cream 1.5g/L; Culture condition: dissolved oxygen amount is 15-18%, stirring velocity 200rpm, 31 ℃ of leavening temperatures;
10% the inoculum size of (3) seed liquor being produced jar substratum inserts to produce and jar cultivates, and produces a jar culture medium prescription and is: Semen Maydis powder 20g/L, soybean cake powder 50g/L, MgSO
47H
2O 0.5g/L, MnSO
4H
2O 10g/L; Culture condition: dissolved oxygen amount is 15-18%, stirring velocity 200rpm, 31 ℃ of leavening temperatures; Being cultured to final living weight is 10
9Cfu/g, the gemma rate of formation reaches more than 97%, and fermentation is accomplished;
(4) after fermentation was accomplished, nutrient solution went out jar, dressed up solid dosage with WHITE CARBON BLACK absorption with packing bag.
Bacterial strain subtilis of the present invention (Bacillus subtilis) from tobacco leaf look unfamiliar the border separation screening to; Through the experiment proof; The fermented liquid of subtilis of the present invention has very strong restraining effect to fungal disease pathogenic bacterias such as Alternaria alternate, pepper anthracnoses; Alternaria alternate, black shank, pepper anthracnose, tomato wilt, cucumber eqpidemic disease, eggplant verticillium wilt, big purple blotch of onion, asparagus stem wilt etc. are demonstrated good protection effect; Simultaneously tobacco, capsicum, cucumber, tomato and eggplant had good growth-promoting functions; Therefore, subtilis of the present invention can be used to prepare the microbial inoculum of biological control tobacco or vegetable fungi disease, also can be used to prepare the growth promoter that promotes tobacco or vegetable growth.In addition, experiment shows that also bacterial strain of the present invention also has effect to the raising of quality of tobacco.Can predict, bacterial strain of the present invention will have broad application prospects in the biological control field of tobacco or vegetable fungi disease.
In the foregoing description, said Alternaria alternate is caused by alternaric bacteria tobacco specialized form (Altternaria altternata f.sp.nicotianae).Said black shank is caused by phytophthora parasitica oronoco (Phytophthora parasitica var.nicotianae).Said capsicum epidemic disease is caused by Phytophthora capsici (Phytophthora capsici).Said pepper anthracnose is caused by capsicum thorn dish spore (Colletotrichum capsici).Said tomato wilt is caused by tomato point sickle spore bacterium tomato specialized form (Fusarium oxysporum f.sp.lycopersici).Said cucumber fusarium axysporum is caused by sharp sickle spore bacterium cucumber specialized form germ (Fusarium oxysporum f.sp.cucumerinum).Said cucumber eqpidemic disease is caused by melon epidemic disease mould (Phytophthora melonis).Said eggplant verticillium wilt is caused by big beautiful Verticillium (Verticillium dahliae).Said big purple blotch of onion is caused by chive chain lattice spore (Alternaria porri).Said asparagus stem wilt is caused by Radix Asparagi Phomopsis (Phomopsis asparagi).
Description of drawings
Subtilis Tpb55 called after Bacillus subtilis of the present invention; Be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on December 29th, 2008; Its deposit number is CGMCCNO.2843; The preservation address is: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City institute of microbiology of the Chinese Academy of Sciences, postcode: 100101.
Fig. 1: subtilis Tpb55 of the present invention (Bacillus subtilis Tpb55) bacterial strain is to the dull and stereotyped restraining effect synoptic diagram of the growth of pepper anthracnose bacterium (Colletotrichum capsici) mycelia.
Fig. 2: subtilis Tpb55 of the present invention (Bacillus subtilis Tpb55) bacterial strain suppresses tobacco brown spot pathogen (Alternaria alternata) synoptic diagram, and visible among the figure, deformity takes place tobacco brown spot pathogen, and mycelia is expanded and is nodositas.
Fig. 3: subtilis Tpb55 of the present invention (Bacillus subtilis Tpb55) bacterial strain is handled tobacco brown spot pathogen (Alternaria alternata) synoptic diagram, and visible among the figure, the mycelia end expands, and breaks, and protoplasma leaks.
Fig. 4: subtilis Tpb55 of the present invention (Bacillus subtilis Tpb55) bacterial strain fermentation liquor is handled tobacco brown spot pathogen (Alternaria alternata) synoptic diagram, and visible among the figure, the brown spot pathogen conidium does not sprout, and produces vesica.
Fig. 5: subtilis Tpb55 of the present invention (Bacillus subtilis Tpb55) bacterial strain fermentation liquor is handled tobacco brown spot pathogen (Alternaria alternata) synoptic diagram, and is visible among the figure, the conidiosporangium follicular rupture, and conidium is thoroughly cleared up.
Fig. 6: conidium normally sprouts tobacco brown spot pathogen (Alternaria alternata), mycelia normal growth synoptic diagram.
Embodiment
Below in conjunction with embodiment the present invention is further described.
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Employed material, reagent all can obtain from commercial sources like no specified otherwise among the following embodiment.
Embodiment 1 bacterial strain is to the restraining effect of tobacco and the growth of vegetable fungi disease pathogenic bacteria
Adopt the filter paper method to measure the antagonistic action of bacterial strain Tpb55: to get fermented liquid 20 μ l on the sterilization filter paper to tobacco and vegetable fungi disease germ; Move to the dull and stereotyped central authorities of oat medium after drying; Equidistant access pathogenic bacteria bacterium cake (Φ 0.5cm) around filter paper; Cultivate 3～4d, mensuration inhibition zone size in 28 ℃.Every kind of target bacterium is repeated results averaged 3 times.
Oat nutrient agar: rolled oats 30g, agar 20g, water 1000mL.Rolled oats adds water 1000mL, on boiling water bath, heats 1h, adds water after the filtered through gauze and supplies 1000mL, add the packing sterilization of agar-agar fusing back (121 ℃, 20min).
The result is as shown in table 1; It is thus clear that bacterial strain Tpb55 has the obvious suppression effect to pathogenic fungies such as tobacco brown spot pathogen, black shank, phytophthora capsici, pepper anthracnose bacterium, tomato wilt bacterium, cucumber fusarium axysporum, cucumber phytophthora, eggplant verticillium wilt bacterium, big purple blotch of onion bacterium, asparagus stem wilt bacterias.Shown in Figure 1 is that the Tpb55 bacterial strain is to pepper anthracnose bacterium mycelial growth restraining effect.Picking inhibition zone edge mycelia microscopically is observed, and discovery Tpb55 bacterial strain can cause pathogenic bacteria mycelia deformity even clear up.As shown in Figure 2, the Tpb55 bacterial strain causes tobacco brown spot pathogen to expand and is nodositas, and the top forms vesicle, after vesicle breaks protoplasma takes place and leaks (Fig. 3).
Table 1 bacterial strain Tpb55 is to the plant pathogenic fungi restraining effect
Embodiment 2 bacterial strains are to the restraining effect of Alternaria alternate pathogenic bacteria spore germination
With the A.alternata spore of Tpb55 crude extract processing, the contrast spore germination rate reaches 60% behind the 6h, does not see sprouting and handle spore, and the contrast spore has been sprouted (shown in Figure 6) above 90% behind the 12h, and processing spore 80% is not sprouted and become deformity.Crude extract begins near the germ tube to expand after handling 24h, forms blister projection (shown in Figure 4), and has inclusion to begin to reveal, to all clearing up (shown in Figure 5).
Embodiment 3 bacterial strains are to the growth-promoting functions of tobacco and vegetables
Preparation Tpb55 bacterial strain fermentation liquor (31 ℃, 180r/m72h).(bacteria containing amount is about 10 with the Tpb55 bacterial strain fermentation liquor for the tobacco that health is full, capsicum, cucumber, eggplant, tomato seeds
8Cfu/mL) after 50 times of diluents soak 12h, be seeded in and a sterilization cubic metre of earth basin be housed (in 160 * 220mm), soaking seed with sterilized water with substratum is contrast, and every kind of plant is handled 3 basins, and every basin is broadcast 15 seeds.After treating that the 4th true leaf of above-mentioned plant launches fully, (bacteria containing amount is about 10 to every basin with the 30mLTpb55 bacterial strain fermentation liquor
8Cfu/mL) 50 times of dilution liquid irrigating roots, the substratum and the sterilized water of respective amount are watered in contrast respectively.Behind the 10d, plant is begun 2-3 true leaf reciprocal from top use portable photosynthetic determinator (Li-6400, the Li-Cor In U.S.) to measure its Net Photosynthetic Rate (Pn).Bacterium liquid is handled back 20d seedling is extracted, and tap water is cleaned and dried, and surveys its fresh weight and plant height, and dry weight is claimed in 80 ℃ of oven dry down of Tpb55 bacterial strain fermentation liquor.
Indoor pot is measured the result and is shown (shown in the table 2); After bacterial strain fermentation liquor is handled; Tobacco, capsicum, cucumber, eggplant, tomato plant Net Photosynthetic Rate, plant height, fresh weight and dry weight all have increase in various degree than sterilized water and substratum, and above-mentioned plant-growth is had obvious facilitation.
Table 2 bacterial strain is to the promoter action of tobacco and vegetable growth
Embodiment 4 bacterial strains are to the greenhouse pot culture control test of tobacco and vegetable fungi disease
In order to check the effect of subtilis Tpb55 control black shank, pepper anthracnose, tomato wilt, cucumber eqpidemic disease, eggplant verticillium wilt, handle the target plant and behind 24h, inoculate phytopathogen with Tpb55.
Particularly, preparation Tpb55 bacterial strain fermentation liquor (31 ℃, 180r/m72h).Tobacco, capsicum, cucumber, eggplant, tomato are planted in the flowerpot of bactericidal nurishing soil, to the 4th leaf period.
Next, every kind of pathogenic agent is inoculated the target plant according to and the following and is infected.With phytophthora parasitica oronoco (Phytopthora parasitica var.nicotianae) zoospore suspension (4 * 10
3Sporocyst/mL) inoculation cigarette seedling brings out black shank,, with capsicum thorn dish spore (Colletotrichum capsici) spore suspension (1 * 10
6Spore/mL) inoculation capsicum seedling induces pepper anthracnose that the zoospore suspension-s (1 * 10 of melon epidemic disease mould (Phytophthora melonis) takes place
3Spore/mL) the inoculation cucumber seedling brings out the cucumber eqpidemic disease, with tomato point sickle spore bacterium tomato specialized form (Fusarium oxysporum f.sp.lycopersici) spore suspension (1 * 10
6Spore/mL) the inoculating tomato seedling is induced tomato wilt, big beautiful Verticillium (Verticillium dahliae) spore suspension (1 * 10
6Spore/mL) inoculate eggplant seedling inducing eggplant verticillium, 28～30 ℃ of heat and moisture preservings induce disease to take place.
The medicament contrast is respectively 400 times of liquid of control black shank master's with medicament 58% metalaxyl-mn-zn; 600 times of liquid of control pepper anthracnose master's with medicament 75% m-tetrachlorophthalodinitrile wettable powder; Prevent and treat 1000 times of liquid of tomato wilt master with medicament 50% carbendazol wettable powder; 600 times of liquid of cucumber blight disease master's with medicament 75% m-tetrachlorophthalodinitrile wettable powder, 600 times of liquid of control eggplant verticillium wilt master's with medicament 70% thiophanate methyl wettable powder
The potted plant test-results of preventing and treating is as shown in table 3, and Tpb55 has preventive effect preferably to black shank, pepper anthracnose, tomato wilt, cucumber eqpidemic disease and eggplant verticillium wilt etc., and control effect and above-mentioned disease master's with medicament are equal to or slightly better.
Table 3 bacterial strain is to the greenhouse control effect test of tobacco and vegetable fungi disease
Embodiment 5 bacterial strains are to the field controling test of tobacco and vegetable fungi disease
Black shank control is tested and was arranged in the sick garden of balck shank, tobacco institute of the Chinese Academy of Agricultural Sciences Jimo experiment base in 2009 and carries out, and practice ground is field, a Plain piece, and soil is the leaching cinnamon soil.June 22 transplanted, line-spacing 100cm, and spacing in the rows 30cm, the cultivation condition of all experiment sub-districts is consistent.Before the transplanting, the dilution of Tpb55 fermented liquid is dipped in root for 200 times, and the balck shank their early stage begins to irritate root, and 200 times of diluent 50mL of Tpb55 fermented liquid are irritated in every strain, respectively at July 23 and July 31 irritating root twice.District's group arrangement at random, every sub-district area 60m are respectively handled in test
2, four repetitions.With 400 times of liquid of 58% metalaxyl-mn-zn is the medicament contrast, is blank with the clear water.Respectively investigate one time disease index, investigation result such as table 4 on July 23, August 11.
Pepper anthracnose control test is selected in capsicum field, crossdrift village, Jimo dragon's fountain town, Qingdao, and soil is the leaching cinnamon soil, and capsicum was transplanted on May 17th, 2010.District's group arrangement is at random respectively handled in test, and every sub-district kind has capsicum 60 strains, 4 repetitions.To 200 times of diluents of blade spraying Tpb55 fermented liquid, began test on July 13 when capsicum begins to fall ill, capsicum is in the best fruiting period during test; Experimental plot piece morbidity is heavier; July 21 sprayed 1 time again, was the medicament contrast with 600 times of liquid of 75% m-tetrachlorophthalodinitrile wettable powder, was blank with the clear water.Respectively investigate one time disease index, investigation result such as table 4 on July 13, August 31.
Tomato wilt, cucumber eqpidemic disease, eggplant verticillium wilt control test all are located at Liu Wang town, Jiaonan City, Qingdao warmhouse booth, and selection is fallen ill serious and the booth of continuous cropping more than 3 years carried out test.
Tomato was transplanted on September 27th, 2010, behind the 7d, and 200 times of dilutions of Tpb55 fermented liquid liquid irrigating root, every strain 50mL.After tomato wilt began morbidity, November 6, November 15, continuous irrigation root twice was the medicament contrast with 1000 times of liquid of 50% carbendazol wettable powder, was blank with the clear water.Every processing repetition 4 times, every sub-district area 64m
2, 100 strains of plantation tomato.November 6, twice disease index of investigation on November 25, investigation result such as table 4.
On March 10th, 2010 was transplanted cucumber, and when beginning to test, cucumber vine has about 2, and eqpidemic disease takes place heavier.Experimental plot area 36m
2, 80 strains of every sub-district plantation cucumber repeat four times.Spraying 200 times of diluents of Tpb55 fermented liquid continuously on April 17,24 days 2 times, is the medicament contrast with 600 times of liquid of 75% m-tetrachlorophthalodinitrile wettable powder, is blank with the clear water.April 17,2 disease indexs of investigation on May 5, investigation result such as table 4.
It is in March, 2010-June that eggplant verticillium wilt is prevented and treated test period.Setting date is March 12, and April 20 began to irritate root at the eggplant their early stage, the every strain 50mL of 200 times of diluents of Tpb55 fermented liquid, and April 27 irritated the 2nd time.With 600 times of liquid of 70% thiophanate methyl wettable powder is the medicament contrast, is blank with the clear water.Sub-district area 10m
2, 20 strains of every sub-district plantation eggplant repeat 4 times.Respectively investigate one time disease index, result such as table 4 on April 20, May 10.
As shown in table 4, Tpb55 has preventive effect preferably to black shank, pepper anthracnose, tomato wilt, cucumber eqpidemic disease and eggplant verticillium wilt etc., and control effect and above-mentioned disease master's with medicament are equal to or slightly better.
Table 4 bacterial strain is to the control effect test of tobacco and vegetable fungi disease
Embodiment 6 bacterial strains are to the Alternaria alternate control effect and to the influence of flue-cured tobacco yield and quality
Alternaria alternate control test arrangement is little one hundred houses vega in the Luliang, Yunnan Province, and flue-cured tobacco cultivars is K326, and the transplanting phase is on April 23rd, 2008, begins dispenser at the initial stage of a disease.Spraying 200 times of diluents of Tpb55 fermented liquid continuously 2 times July 23, August 30, is the medicament contrast with 500 times of liquid of 40% dimetachlone wettable powder, is blank with the clear water.Sub-district area 66.7m
2, cigarette 100 strains are planted in every sub-district.Respectively investigate one time disease index July 23, August 9.As shown in table 5, Tpb55 has good control effect to Alternaria alternate, and 200 times of liquid control effect of fermented liquid can reach more than 70%, are superior to producing main with medicament dimetachlone.
Table 5 bacterial strain is to the field control effect of Alternaria alternate
The ripe tobacco leaf of all processing is gathered respectively by the sub-district and is toasted, and statistics cured tobacco leaf output, by the national standard classification, confirms tobacco leaf ratios such as upper, middle and lower.Each carries out chemical composition analysis after handling and selecting for use the B2F tobacco sample to grind after the tobacco flue-curing, plant appearance through the H2SO4-H2O2 wet ashing technique disappear boil after, measure nitrogen content with nitrogen determination, use flame photometry] measure potassium content; Measure total reducing sugar and reducing sugar content with copper reduction-direct titrimetric method; Measure nicotine content with ultraviolet spectrophotometer method; Measure cl content with Mohr's method.Test-results shows, use the Tpb55 bacterial strain fermentation liquor after, yield of tobacco, go up medium grade cigarette ratio, first-class cigarette ratio and the output value raising (table 6) in various degree all arranged.
Table 6 is used the influence of bacterial strain fermentation liquor to the tobacco leaf economic characters
As shown in table 7, it is little to the inherent chemical composition influence of upper tobacco leaf to use the Tpb55 bacterial strain fermentation liquor, shows that mainly reducing sugar, potassium have a little raising, and the Cl ion content reduces, and nitrogen base ratio, sugared alkali ratio, sugared nitrogen ratio, potassium chlorine are than equal size all in the reasonable scope.
Table 7 is used the influence of bacterial strain fermentation liquor to the inherent chemical ingredients of upper tobacco leaf B2F
After using the Tpb55 bacterial strain fermentation liquor, upper tobacco leaf sensory evaluating smoking quality improves to some extent, and is mainly reflected in fragrance matter, perfume quantity raising, and assorted gas and pungency reduce (table 8)
Table 8 is used the influence of bacterial strain fermentation liquor to upper tobacco leaf B2F sensory evaluating smoking index
Embodiment 7 preparation microbial preparations
Be made up of the full nutrient solution culture of subtilis Bacillus subtilis Tpb55 and the spore of subtilis Bacillus subtilis Tpb55, the preparation method is following:
(1) subtilis Bacillus subtilis Tpb55 test tube kind is inoculated in broth culture and shakes in the bottle, shaking culture is to logarithmic phase;
(2) with the volume ratio 10% inoculum size access 30L seeding tank of above-mentioned cultured bacterial classification by the seed tank culture base, be cultured to logarithmic phase, the used culture medium prescription of seeding tank is: glucose 10g/L, peptone 3g/L, MgSO
40.5g/L, yeast extract paste 1.5g/L, Carnis Bovis seu Bubali cream 1.5g/L; Culture condition: dissolved oxygen amount is 15-18%, stirring velocity 200rpm, 31 ℃ of leavening temperatures, incubation time 72h;
10% the inoculum size of (3) seed liquor being produced jar substratum inserts to produce and jar cultivates, and produces a jar culture medium prescription and is: Semen Maydis powder 20g/L, soybean cake powder 50g/L, MgSO
47H
2O 0.5g/L, MnSO
4H
2O 10g/L; Culture condition: dissolved oxygen amount is 15-18%, stirring velocity 200rpm, 31 ℃ of leavening temperatures, incubation time 72h; After the cultivation, final living weight is 10
9Cfu/g, the gemma rate of formation reaches more than 97%, and fermentation is accomplished;
(4) after fermentation was accomplished, nutrient solution went out jar, dressed up solid dosage with WHITE CARBON BLACK absorption with packing bag.
Claims (5)
1. the application of subtilis in the vegetable fungi disease control, it is characterized in that: said vegetable fungi disease comprises: capsicum epidemic disease, pepper anthracnose, cucumber fusarium axysporum, eggplant verticillium wilt, big purple blotch of onion, asparagus stem wilt; Said subtilis called after: subtilis (Bacillus subtilis), carry out preservation on December 29th, 2008 by China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.2843.
2. the application of subtilis in promoting vegetable growth; It is characterized in that: said subtilis called after: subtilis Bacillus subtilis; Carry out preservation on December 29th, 2008 by China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.2843.
3. application according to claim 2 is characterized in that: said vegetables comprise: cucumber, capsicum, eggplant, tomato.
4. the application of subtilis in improving quality of tobacco; It is characterized in that: said subtilis called after: subtilis Bacillus subtilis; Carry out preservation on December 29th, 2008 by China Committee for Culture Collection of Microorganisms common micro-organisms center, deposit number is CGMCC No.2843.
5. microbial preparation; It is characterized in that: form by the full nutrient solution culture of subtilis Bacillus subtilis Tpb55 and the spore of subtilis Bacillus subtilis Tpb55; Said subtilis carries out preservation on December 29th, 2008 by China Committee for Culture Collection of Microorganisms common micro-organisms center, and deposit number is CGMCC No.2843;
Said microbial preparation prepares through following preparation method:
(1) subtilis Bacillus subtilis Tpb55 test tube kind is inoculated in broth culture and shakes in the bottle, shaking culture is to logarithmic phase;
(2) with the volume ratio 10% inoculum size access 30L seeding tank of above-mentioned cultured bacterial classification by the seed tank culture base, be cultured to logarithmic phase, the used culture medium prescription of seeding tank is: glucose 10g/L, peptone 3g/L, MgSO
40.5g/L, yeast extract paste 1.5g/L, Carnis Bovis seu Bubali cream 1.5g/L; Culture condition: dissolved oxygen amount is 15-18%, stirring velocity 200rpm, 31 ℃ of leavening temperatures;
10% the inoculum size of (3) seed liquor being produced jar substratum inserts to produce and jar cultivates, and produces a jar culture medium prescription and is: Semen Maydis powder 20g/L, soybean cake powder 50g/L, MgSO
47H
2O 0.5g/L, MnSO
4H
2O 10g/L; Culture condition: dissolved oxygen amount is 15-18%, stirring velocity 200rpm, 31 ℃ of leavening temperatures; Being cultured to final living weight is 10
9Cfu/g, the gemma rate of formation reaches more than 97%, and fermentation is accomplished;
(4) after fermentation was accomplished, nutrient solution went out jar, dressed up solid dosage with WHITE CARBON BLACK absorption with packing bag.
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