CN102676444B - Culture medium for promoting growth of bacillus and application thereof - Google Patents
Culture medium for promoting growth of bacillus and application thereof Download PDFInfo
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Abstract
The invention discloses a culture medium for promoting growth of bacillus and application thereof. According to the culture medium, K+ is added into a Tryptone Broth culture medium until the final concentration is 2.2mM, and Cu2+ is added until the final concentration is 8 mu M. K2HPO4 at the final concentration of 0.3g/L and CuSO4.5H2O at the final concentration of 0.002g/L are added into the culture medium. Experiments prove that when the culture medium is used for culturing Bacillus subtilis B201 CGMCC No.5786, the bacteria content of a fermentation liquor can be improved double, and the spore forming time is shortened by 15 percent. Therefore, the production cost is reduced by 30 percent. The culture medium has high promotion value. The invention provides a reference for the optimization of a Bacillus subtilis fermentation culture medium, and provides theoretical guidance for the research and development of related biocontrol agents.
Description
Technical field
The present invention relates to a kind of substratum and application thereof that promotes the genus bacillus growth, particularly substratum and the application thereof of the interpolation mineral nutriment of a kind of promotion subtilis (Bacillus subtilis) growth.
Background technology
Genus bacillus (Bacillus sp.), due to the gemma that can produce resistance, can be stood multiple poor environment, has the effects such as short length, diseases prevention, is a kind of valuable source of biotechnological formulation.Mineral nutriment is the requisite class nutritive substance of microorganism growth, mainly participate in cellularstructure and form, and with energy, shift, cell permeability is regulated relevantly, is active group component or the activator of enzyme.But add and excessively also can poison microorganisms.
At present both at home and abroad the fermentation of bacillus study hotspot is to take the screening of the carbon nitrogen source that meta-bolites is target and the optimization of culture condition, lacks to take the correlative study that viable bacteria is target product, and mineral nutriment it be unclear that the impact of genus bacillus growth velocity.
Summary of the invention
The purpose of this invention is to provide a kind of substratum and application thereof that promotes the genus bacillus growth.
The substratum of cultivation genus bacillus provided by the present invention (Bacillus sp.) is to add K in Tryptone Broth substratum
+to its final concentration be 2.2-5.1mM, as 2.2mM, add Cu simultaneously
2+to its final concentration substratum that is 8 μ M.
In the present invention, the solute of described Tryptone Broth substratum is Tryptones, sucrose and NaCl, and solvent is water; The final concentration of described Tryptones in described Tryptone Broth substratum is 10g/L, the final concentration of described sucrose in described Tryptone Broth substratum is 10g/L, and the final concentration of described NaCl in described Tryptone Broth substratum is 5g/L; The pH of described Tryptone Broth substratum is 7.5.
Described K
+can derive from K
2hPO
4, KH
2pO
4, KCl, K
2sO
4, KOH or K
2cO
3etc. multiple potassium-containing compound, in one embodiment of the invention, described K
+specifically derive from K
2hPO
4, certain K
2hPO
4hydrate also can reach identical effect.
Described Cu
2+can derive from CuSO
45H
2o, CuCl
2deng, in one embodiment of the invention, described Cu
2+specifically derive from CuSO
45H
2o, CuSO certainly
4other hydrates and CuSO
4also can reach identical effect.
Described substratum is storage type substratum (powder) or instant substratum (solution); Described storage type substratum is by K
2hPO
4, CuSO
45H
2o(or CuSO
4), Tryptones, sucrose and NaCl be according to 0.19g:0.002g(or 0.00128g): the proportioning of 10g:10g:5g forms; The solvent of described instant substratum is water, and solute is K
2hPO
4, CuSO
45H
2o, Tryptones, sucrose and NaCl, in described instant substratum, described K
2hPO
4final concentration be 0.19g/L, described CuSO
45H
2the final concentration of O is 0.002g/L, and the final concentration of described Tryptones is 10g/L, and the final concentration of described sucrose is 10g/L, and the final concentration of described NaCl is 5g/L, pH7.5.
In one embodiment of the invention, described substratum is specially above-mentioned instant substratum.
In following (1)-(4), any application also belongs to protection scope of the present invention to described substratum:
(1) improve genus bacillus (Bacillus sp.) growth velocity;
(2) improve genus bacillus (Bacillus sp.) output;
(3) shorten genus bacillus (Bacillus sp.) the sporulation time;
(4) improve genus bacillus (Bacillus sp.) spore forming rate.
In the present invention, described genus bacillus (Bacillus sp.) growth velocity is embodied in described genus bacillus (Bacillus sp.) thalli growth curve (OD
600) slope on, slope is larger, described genus bacillus (Bacillus sp.) growth velocity is faster.Described genus bacillus (Bacillus sp.) output is embodied in described genus bacillus (Bacillus sp.) thalli growth curve (OD
600) the corresponding OD of point (X-coordinate) sometime
600value (ordinate zou) is upper, OD
600be worth greatlyr, the output of this time point of described genus bacillus (Bacillus sp.) is higher.Described genus bacillus (Bacillus sp.) the sporulation time is embodied in fermentor tank thalli growth curve (OD
600) time point of end, curve is shorter, and the time that forms gemma is shorter (described growth curve (OD just
600) the corresponding microscope inspection spore forming rate of time point of end reaches for 80% time).Described genus bacillus (Bacillus sp.) spore forming rate is embodied in 100 times of observed data records of microscope, with the rear micro-Microscopic observation that dyes, calculates, and in a visual field, the quantity of gemma in 100 cells.Spore forming rate=100% * (quantity of gemma/100).
A further object of the present invention is to provide the method for a kind of cultivation genus bacillus (Bacillus sp.).
The method comprises described genus bacillus (Bacillus sp.) is inoculated in above-mentioned substratum, at 30-32 ℃, as 32 ℃ of steps of carrying out shaking culture or aerated culture.
In aforesaid method, while cultivating in the supporting culture plate of growth curve analyser, the time of described cultivation is 7-64h.Be specially in an embodiment of the present invention 20h, or 64h.
In aforesaid method, while cultivating in fermentor tank, the time of described cultivation can be 34-64h, is specially in one embodiment of the invention 40h.Air flow in described culturing process can be 0.5 (V/Vmin) to 1.5 (V/Vmin); Described air flow recently means with the volume of air by the unit volume nutrient solution in per minute.
In one embodiment of the invention, during fermentation culture, it is 0.5 (V/Vmin) that air flow in described culturing process is specially and cultivates early stage (starting to before the thalli growth logarithmic phase from cultivation) air flow, late stage of culture (finish from the thalli growth logarithmic phase to the maximum growth amount phase) 1.5 (V/Vmin) (described air flow recently means with the volume of air by the unit volume nutrient solution in per minute, and the volume that 0.5 (V/Vmin) means to pass in per minute gas is half of nutrient solution volume).
In an embodiment of the present invention, above-mentioned all described genus bacillus (Bacillus sp.) are subtilis (Bacillus subtilis), are specially subtilis (Bacillus subtilis) B201 CGMCC No.5786.
Another object of the present invention is to provide the substratum mineral nutriment of a kind of cultivation genus bacillus (Bacillus sp.)
Substratum mineral nutriment provided by the present invention, the K that is specifically 95g:1g by mass ratio
2hPO
4and CuSO
45H
2o forms.Certainly also can be by CuSO
45H
2o is converted to the CuSO of respective quality
4.In use, described K
2hPO
4final concentration in described substratum is 0.19g/L, described CuSO
45H
2the final concentration of O in described substratum is 0.002g/L.
Described substratum mineral nutriment composition is at the above-mentioned substratum of preparation, or the application of cultivating in genus bacillus (Bacillus sp.) also belongs to protection scope of the present invention.
The present invention is the impact on the genus bacillus growth velocity by research mineral nutriment, clearly affect the main mineral nutriment of genus bacillus (Bacillus sp.) growth, experimental results show that, use Tryptone Broth culture medium culturing subtilis (Bacillus subtilis) the B201 CGMCC No.5786 that adds this mineral nutriment, the fermented liquid bacteria containing amount can improve approximately 2 times, sporulation time shorten 15%.So, production cost will reduce by 30%.There is good promotional value.The present invention provides reference for the optimization of fermentation of bacillus substratum, and development and the exploitation for relevant biological prevention and control agent simultaneously provides theoretical direction.
Preservation proves
The biomaterial of ginseng certificate: B201
The Classification And Nomenclature of suggestion: subtilis (Bacillus subtilis)
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is called for short: CGMCC
Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City
Preservation date: on February 22nd, 2012
The preservation center numbering of registering on the books: CGMCC No.5786
The accompanying drawing explanation
The impact of the various mineral nutriment that Fig. 1 is different concns on subtilis (Bacillus subtilis) B201 CGMCC No.5786 growth.Wherein, A represents K(KH
2pO
4); B represents Mg(MgSO
47H
2o); C represents Mn(MnSO
4h
2o); D represents Fe(FeSO
47H
2o); E represents Zn(ZnSO
47H
2o); F represents Cu(CuSO
45H
2o); G represents Mo(Na
2moO
42H
2o); H represents Ca(CaCl
2).
The impact of K ion pair subtilis (Bacillus subtilis) the B201 CGMCC No.5786 growth that Fig. 2 is different sources.
Fig. 3 contains optimum mineral nutriment proportioning and the comparison diagram of the growth effect containing the Tryptone Broth substratum of mineral nutriment, subtilis (Bacillus subtilis) B201 CGMCC No.5786 not cultivated in the growth curve analyser.Wherein, TS means Tryptone Broth substratum.
Fig. 4 contains optimum mineral nutriment proportioning and the comparison diagram of the growth effect containing the Tryptone Broth substratum of mineral nutriment, subtilis (Bacillus subtilis) B201 CGMCC No.5786 not cultivated in the 50L fermentor tank.Wherein, TS means Tryptone Broth substratum.
The comparison diagram that Fig. 5 is the industrial fermentation substratum that contains different concns K ion growth effect that subtilis (Bacillus subtilis) B201 CGMCC No.5786 is cultivated in the 300mL triangular flask.Wherein, a is not for adding the industrial fermentation substratum of external source K ion; B has added the KH that final concentration is 0.3g/L
2pO
4the industrial fermentation substratum; C has added the KH that final concentration is 0.5g/L
2pO
4the industrial fermentation substratum; D has added the KH that final concentration is 0.7g/L
2pO
4the industrial fermentation substratum.
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The LB liquid nutrient medium: the yeast extract that the NaCl that the Tryptones that final concentration is 10g/L, final concentration are 10g/L, final concentration are 5g/L, surplus are water, pH7.0-7.2.
LB solid medium: separately add the agar powder that final concentration is 15g/L on the basis of LB liquid nutrient medium.
Tryptone Broth liquid nutrient medium: the NaCl that the sucrose that the Tryptones that final concentration is 10g/L, final concentration are 10g/L, final concentration are 5g/L, surplus are water, pH7.5.
Tryptone Broth solid medium: separately add the agar powder that final concentration is 15g/L on the basis of Tryptone Broth liquid nutrient medium.
Tryptose soya agar (Tryptic Soy Agar, TSA) flat board: the Tryptones that final concentration is 15g/L, the soy peptone that final concentration is 5g/L, the NaCl that final concentration is 5g/L, surplus is water, pH7.5.
In following embodiment, the preparation of related seed liquor is all carried out as follows:
With tryptose soya agar (Tryptic Soy Agar, TSA) dull and stereotyped activation subtilis (Bacillus subtilis) B201 CGMCC No.5786 bacterial classification, after 30 ℃ of incubated overnight, picking list bacterium colony be inoculated in 15mL TS liquid nutrient medium 32 ℃, 160r/min incubated overnight (12h) from flat board, correct through the microscopy thalli morphology, pollution-free, by cultured bacterium liquid, the centrifugal 5min of 6000r/min, by sterile water wash twice, make bacteria suspension.Survey the OD value with spectrophotometer 600nm wavelength, make the OD value reach 1, obtain the seed liquor of subtilis (Bacillus subtilis) B201 CGMCC No.5786.
Determining of the substratum mineral nutriment used of embodiment 1, the growth of promotion genus bacillus
One, mineral nutriment basic concentration scope determines
1, the preparation of the preparation of different concns mineral nutriment and corresponding substratum
Because the quality of the needed mineral nutriment of every processing is very little, weighing error is large, so first prepare mother liquor, mother liquor requires solute to dissolve fully, according to the required volume added of the every processing of the concentration conversion of mother liquor, the increase of this volume should be ignored whole for the impact of culture volume; Content for the mineral nutriment of itself in Tryptone Broth substratum has carried out detecting (content of each element in Tryptone Broth substratum is in Table 1) with atomic absorption spectrophotometer, according in " general microbiology ", the general concentration range of required each mineral nutriment of microorganism growth, set the corresponding concentration gradient of each mineral nutriment, as shown in table 1.
Table 1 mineral nutriment concentration is set
Annotate: " setting concentration " refers to the final concentration of the respective compound that contains each mineral nutriment (i.e. source compound) in substratum that external source adds.
Take Tryptone Broth substratum as basis, add respectively 8 kinds of mineral nutriment of respective settings concentration, preparation obtains containing in 8 kinds of mineral nutriment any, and the final concentration of the compound that contains corresponding mineral nutriment added (in table 1, carrying out source compound) in substratum is any different culture media in three concentration gradients in " setting concentration " hurdle.
2, mineral nutriment basic concentration scope determines
Adopt the Tryptone Broth substratum that contains different concns simple ore matter nutriment of preparing in step 1, by single single-factor method, detect the impact of the mineral nutriment of different concns on the growth of subtilis (Bacillus subtilis) B201CGMCC No.5786.Concrete operations are as follows:
In the Tryptone Broth substratum that contains different concns simple ore matter nutriment that the seed liquor of subtilis (Bacillus subtilis) B201 CGMCC No.5786 is inoculated in step 1 respectively to be prepared with the inoculum size of volumn concentration 2%, vibrate 2 minutes, add growth curve analyser (bioscreen, Finland, Oy Growth Curves Ab Ltd.) in supporting culture plate, every hole adds the nutrient solution that volume is 150 μ L, every processing repeats for 3 times, and the culture plate of application of sample is put into to the growth curve analyser, and 32 ℃, after shaking 64h continuously, every 15min surveys an OD value.Result is got the mean value repeated 3 times.The Tryptone Broth substratum (CK) in contrast do not add any mineral nutriment is set simultaneously.
As shown in Figure 1, these 8 kinds of mineral nutriment are all influential to the bacterium amount of subtilis (Bacillus subtilis) B201 CGMCC No.5786 as can be seen from Figure 1 for result, and the interpolation of K ion, promote its growth, but the high density (KH of 4g/L
2pO
4) can suppress the growth of bacterium; Its growth effect of Zn ion pair is not obvious; Other 6 kinds of mineral nutriment just have and affect the growth of bacterium.
Two, mineral nutriment is to the dominant factor of subtilis growth effect and determining of consumption thereof
(1) according to the concrete concentration of 5 corresponding various mineral nutriment of level shown in table 2 in corresponding substratum, and the required Tryptone Broth substratum that contains mineral nutriment is processed in 50 of the preparations of the orthogonal design scheme shown in table 3.
(2) by the seed liquor of subtilis (Bacillus subtilis) B201 CGMCC No.5786, the inoculum size with volumn concentration 2% is inoculated into respectively in 50 kinds of Tryptone Broth substratum that contain 8 kinds of mineral nutriment of different concns being prepared in step (1), vibrate 2 minutes, add growth curve analyser (bioscreen, Finland, Oy Growth Curves Ab Ltd.) in supporting culture plate, every hole adds the nutrient solution that volume is 150 μ L, every processing repeats for 3 times, and the culture plate of application of sample is put into to the growth curve analyser, and 32 ℃, 20h is cultivated in concussion, within every 15 minutes, surveys an OD
600value, its growth conditions of on-line monitoring is drawn growth curve.
Each mineral nutriment concentration orthogonal table gauge outfit of table 2 L
50(5
11) unit: g/L
Annotate: concentration (g/L) refers to the final concentrations of each element place compound (in corresponding table 1, carrying out source compound) in substratum such as K, Mg.
The quadrature result is as shown in table 3, wherein, repeats 1, repeats 2 and to repeat the value shown in 3 be OD
600value; Tt repeats OD 3 times
600value add and; Yt repeats OD 3 times
600the mean value of value; T1-T5 mean respectively 5 horizontal Tt values adding and; 220.38 of black matrix mark means to repeat the OD that surveys to some extent 3 times
600the summation of value is 220.38, is denoted as T; SS means to repeat OD 3 times
600the mean value of the sum of square of deviations of value, its formula is SS=1/3*[1/10* (T1
2+ T2
2+ T3
2+ T4
2+ T5
2)-(T
2/ n)], wherein n is for processing number, and n is specially 50 in this embodiment.
The results of analysis of variance is as table 4, and as known from Table 4, with regard to single factors, the K factorial effect is extremely remarkable, and Fe, Cu, Mo factorial effect are remarkable, and other factors are all not remarkable.Result is affected to the variation that inapparent theory of factor open fire is flat little on the result impact, so consider from economic angle, these factors are removed, retain four dominant factors, i.e. K, Fe, Cu, Mo.
Table 3 L50 (
5 11) orthogonal design scheme and mineral nutriment is to the test-results of withered grass gemma growth effect
| Process | K | Mg | Mn | Fe | Zn | Cu | Mo | Ca | Blank 1 | Blank 2 | Blank 3 | Repeat 1 | Repeat 2 | Repeat 3 | Tt | Yt |
| 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 0.940 | 1.128 | 1.134 | 3.20 | 1.07 |
| 2 | 1 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 1.118 | 1.145 | 1.124 | 3.39 | 1.13 |
| 3 | 1 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 3 | 0.978 | 0.968 | 0.953 | 2.90 | 0.97 |
| 4 | 1 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 4 | 0.829 | 1.058 | 1.110 | 3.00 | 1.00 |
| 5 | 1 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 5 | 1.195 | 1.235 | 1.214 | 3.64 | 1.21 |
| 6 | 2 | 1 | 2 | 3 | 4 | 5 | 1 | 2 | 3 | 4 | 5 | 1.601 | 1.624 | 1.567 | 4.79 | 1.60 |
| 7 | 2 | 2 | 3 | 4 | 5 | 1 | 2 | 3 | 4 | 5 | 1 | 1.713 | 1.593 | 1.579 | 4.89 | 1.63 |
| 8 | 2 | 3 | 4 | 5 | 1 | 2 | 3 | 3 | 5 | 1 | 2 | 1.557 | 1.581 | 1.647 | 4.79 | 1.60 |
| 9 | 2 | 4 | 5 | 1 | 2 | 3 | 4 | 5 | 1 | 2 | 3 | 1.618 | 1.633 | 1.621 | 4.87 | 1.62 |
| 10 | 2 | 5 | 1 | 2 | 3 | 4 | 5 | 1 | 2 | 3 | 4 | 1.612 | 1.645 | 1.615 | 4.87 | 1.62 |
| 11 | 3 | 1 | 3 | 5 | 2 | 4 | 4 | 1 | 3 | 5 | 2 | 1.682 | 1.827 | 1.651 | 5.16 | 1.72 |
| 12 | 3 | 2 | 4 | 1 | 3 | 5 | 5 | 2 | 4 | 1 | 3 | 1.706 | 1.730 | 1.746 | 5.18 | 1.73 |
| 13 | 3 | 3 | 5 | 2 | 4 | 1 | 1 | 3 | 5 | 2 | 4 | 1.588 | 1.330 | 1.337 | 4.26 | 1.42 |
| 14 | 3 | 4 | 1 | 3 | 5 | 2 | 2 | 4 | 1 | 3 | 5 | 1.408 | 1.307 | 1.332 | 4.05 | 1.35 |
| 15 | 3 | 5 | 2 | 4 | 1 | 3 | 3 | 5 | 2 | 4 | 1 | 1.770 | 1.663 | 1.510 | 4.94 | 1.65 |
| 16 | 4 | 1 | 4 | 2 | 5 | 3 | 5 | 3 | 1 | 4 | 2 | 1.462 | 1.422 | 1.399 | 4.28 | 1.43 |
| 17 | 4 | 2 | 5 | 3 | 1 | 4 | 1 | 4 | 2 | 5 | 3 | 1.652 | 1.590 | 1.671 | 4.91 | 1.64 |
| 18 | 4 | 3 | 1 | 4 | 2 | 5 | 2 | 5 | 3 | 1 | 4 | 1.555 | 1.563 | 1.626 | 4.74 | 1.58 |
| 19 | 4 | 4 | 2 | 5 | 3 | 1 | 3 | 1 | 4 | 2 | 5 | 1.611 | 1.468 | 1.396 | 4.48 | 1.49 |
| 20 | 4 | 5 | 3 | 1 | 4 | 2 | 4 | 2 | 5 | 3 | 1 | 1.639 | 1.697 | 1.688 | 5.02 | 1.67 |
| 21 | 5 | 1 | 5 | 4 | 3 | 2 | 4 | 3 | 2 | 1 | 5 | 1.602 | 1.754 | 1.486 | 4.84 | 1.61 |
| 22 | 5 | 2 | 1 | 5 | 4 | 3 | 5 | 4 | 3 | 2 | 1 | 1.638 | 1.549 | 1.453 | 4.64 | 1.55 |
| 23 | 5 | 3 | 2 | 1 | 5 | 4 | 1 | 5 | 4 | 3 | 2 | 1.646 | 1.429 | 1.666 | 4.74 | 1.58 |
| 24 | 5 | 4 | 3 | 2 | 1 | 5 | 2 | 1 | 5 | 4 | 3 | 1.573 | 1.722 | 1.758 | 5.05 | 1.68 |
| 25 | 5 | 5 | 4 | 3 | 2 | 1 | 3 | 1 | 5 | 4 | 1.559 | 1.565 | 1.578 | 4.70 | 1.57 | |
| 26 | 1 | 1 | 1 | 4 | 5 | 4 | 3 | 2 | 5 | 2 | 3 | 1.149 | 1.015 | 1.041 | 3.21 | 1.07 |
| 27 | 1 | 2 | 2 | 5 | 1 | 5 | 4 | 3 | 1 | 3 | 4 | 1.086 | 0.977 | 0.993 | 3.06 | 1.02 |
| 28 | 1 | 3 | 3 | 1 | 2 | 1 | 5 | 4 | 2 | 4 | 5 | 1.066 | 1.142 | 1.143 | 3.35 | 1.12 |
| 29 | 1 | 4 | 4 | 2 | 3 | 2 | 1 | 5 | 3 | 5 | 1 | 1.150 | 1.214 | 1.172 | 3.54 | 1.18 |
| 30 | 1 | 5 | 5 | 3 | 4 | 3 | 2 | 1 | 4 | 1 | 2 | 1.126 | 1.131 | 1.105 | 3.36 | 1.12 |
| 31 | 2 | 1 | 2 | 1 | 3 | 3 | 2 | 4 | 5 | 5 | 4 | 1.377 | 1.440 | 1.324 | 4.14 | 1.38 |
| 32 | 2 | 2 | 3 | 2 | 4 | 4 | 3 | 5 | 1 | 1 | 5 | 1.590 | 1.618 | 1.615 | 4.82 | 1.61 |
| 33 | 2 | 3 | 4 | 3 | 5 | 5 | 4 | 1 | 2 | 2 | 1 | 1.631 | 1.670 | 1.609 | 4.91 | 1.64 |
| 34 | 2 | 4 | 5 | 4 | 1 | 1 | 5 | 2 | 3 | 3 | 2 | 0.011 | 1.570 | 1.604 | 3.19 | 1.06 |
| 35 | 2 | 5 | 1 | 5 | 2 | 2 | 1 | 3 | 4 | 4 | 3 | 1.296 | 1.261 | 1.400 | 3.96 | 1.32 |
| 36 | 3 | 1 | 3 | 3 | 1 | 2 | 5 | 5 | 4 | 2 | 4 | 1.646 | 1.668 | 1.674 | 4.99 | 1.66 |
| 37 | 3 | 2 | 4 | 4 | 2 | 3 | 1 | 1 | 5 | 3 | 5 | 0.000 | 1.618 | 1.656 | 3.27 | 1.09 |
| 38 | 3 | 3 | 5 | 5 | 3 | 4 | 2 | 2 | 1 | 4 | 1 | 1.724 | 1.640 | 1.604 | 4.97 | 1.66 |
| 39 | 3 | 4 | 1 | 1 | 4 | 5 | 3 | 3 | 2 | 5 | 2 | 1.733 | 1.742 | 1.717 | 5.19 | 1.73 |
| 40 | 3 | 5 | 2 | 2 | 5 | 1 | 4 | 4 | 3 | 1 | 3 | 1.649 | 1.675 | 1.613 | 4.94 | 1.65 |
| 41 | 4 | 1 | 4 | 5 | 4 | 1 | 2 | 5 | 2 | 3 | 3 | 1.739 | 1.331 | 1.466 | 4.54 | 1.51 |
| 42 | 4 | 2 | 5 | 1 | 5 | 2 | 3 | 1 | 3 | 4 | 4 | 1.744 | 1.767 | 1.756 | 5.27 | 1.76 |
| 43 | 4 | 3 | 1 | 2 | 1 | 3 | 4 | 2 | 4 | 5 | 5 | 1.669 | 1.699 | 1.748 | 5.12 | 1.71 |
| 44 | 4 | 4 | 2 | 3 | 2 | 4 | 5 | 3 | 5 | 1 | 1 | 1.716 | 1.751 | 1.730 | 5.20 | 1.73 |
| 45 | 4 | 5 | 3 | 4 | 3 | 5 | 1 | 4 | 1 | 2 | 2 | 1.441 | 1.242 | 1.224 | 3.91 | 1.30 |
| 46 | 5 | 1 | 5 | 2 | 2 | 5 | 3 | 4 | 4 | 3 | 1 | 1.668 | 1.658 | 1.676 | 5.00 | 1.67 |
| 47 | 5 | 2 | 1 | 3 | 3 | 1 | 4 | 5 | 5 | 4 | 2 | 1.528 | 1.625 | 1.585 | 4.74 | 1.58 |
| 48 | 5 | 3 | 2 | 4 | 4 | 2 | 5 | 1 | 1 | 5 | 3 | 1.670 | 1.787 | 1.756 | 5.21 | 1.74 |
| 49 | 5 | 4 | 3 | 5 | 5 | 3 | 1 | 2 | 2 | 1 | 4 | 1.293 | 1.246 | 1.379 | 3.92 | 1.31 |
| 50 | 5 | 5 | 4 | 1 | 1 | 4 | 2 | 3 | 3 | 2 | 5 | 1.872 | 1.675 | 1.716 | 5.26 | 1.75 |
| T1 | 32.64 | 44.15 | 43.71 | 46.24 | 44.50 | 42.27 | 40.50 | 44.79 | 43.07 | 44.99 | 46.31 | 71.826 | 74.388 | 74.167 | 220.38 | 1.47 |
| T2 | 45.22 | 44.17 | 44.88 | 45.26 | 43.65 | 45.05 | 44.39 | 43.48 | 44.86 | 43.90 | 42.74 | C | 323.79 | |||
| T3 | 46.95 | 44.98 | 44.01 | 44.55 | 43.56 | 41.45 | 45.29 | 43.83 | 44.42 | 40.64 | 44.77 | SST | 12.99 | |||
| T4 | 47.46 | 42.47 | 43.47 | 41.20 | 44.83 | 46.14 | 45.65 | 42.72 | 44.71 | 44.35 | 42.94 | SSR | 0.08 | |||
| T5 | 48.11 | 44.61 | 44.31 | 43.14 | 43.84 | 45.48 | 44.56 | 45.57 | 43.32 | 46.50 | 43.63 | SSt | 9.00 | |||
| SS | 5.60 | 0.12 | 0.04 | 0.52 | 0.04 | 0.58 | 0.57 | 0.17 | 0.09 | 0.62 | 0.29 | SSe | 3.91 |
The variance analysis of table 4 mineral nutriment to the subtilis growth effect
Annotate: * * mean difference extremely significantly (the F value>F
0.01), * mean significant difference (the F value>F
0.05).
Then, the factor main effect is carried out to multiple comparisons, the factor main effect refers to the difference between each factor different levels of table 3.At first calculate each level of factor standard error of the mean, calculate accordingly to obtain minimum significance extreme difference, in Table 5.Obtain the result of multiple comparisons of each factor main effect according to table 5, in Table 6.As can be drawn from Table 6, the best of breed of K, Fe, these four factors of Cu, Mo is K5Fe1Cu4Mo5, according to significant difference analysis between the level of these dominant factors, from economic principle, considers, the best of breed of these four dominant factors is K3Fe1Cu2Mo1, i.e. KH
2pO
4addition in Tryptone Broth substratum is 0.3g/L(level 3, calculates K
+final concentration in Tryptone Broth substratum is 2.2mM), CuSO
45H
2the addition of O in Tryptone Broth substratum is 0.002g/L(level 2, calculates Cu
2+final concentration in Tryptone Broth substratum is 8 μ M), Fe and Mo do not add (level 1).
The minimum significantly extreme difference of table 5 factorial effect multiple comparisons
Table 6 each factor main effect and the result of multiple comparisons
| Level | K | Level | Fe | Level | | Level | Mo | |
| 5 | 48.112a | 1 | 46.235a | 4 | 46.139a | 5 | 45.565a | |
| 4 | 47.462a | 2 | 45.264a | 5 | 45.482a | 1 | 44.788a | |
| 3 | 46.946a | 3 | 44.548a | 2 | 45.046 |
3 | 43.829a | |
| 2 | 45.222b | 5 | 43.139b | 1 | 42.266b | 2 | 43.479a | |
| 1 | 32.639c | 4 | 41.195b | 3 | 41.448b | 4 | 42.72b |
Annotate: different lowercase alphabet differentials different significantly (α=0.01)
His-and-hers watches 3 gained orthogonal experiments (repeat 1, repeat 2 and repeat 3) adopt the DPS analysis software, carry out the multiple comparisons (LSR method) of different treatment, result is as shown in table 7, as shown in Table 7, process 42(K4, Mg2, Mn5, Fe1, Zn5, Cu2, Mo3, Ca1), process 50(K5, Mg5, Mn4, Fe1, Zn1, Cu4, Mo2, Ca3) with to subtilis growth effect maximum, but difference is not remarkable each other.Four dominant factor proportionings are respectively K4Fe1Cu2Mo3, K5Fe1Cu4Mo2, this with there is no significant difference with single factor analysis gained optimum combination K3Fe1Cu2Mo1, as known from Table 6, there is no significant difference between 3,4,5 liang of levels of K, there is no significant difference between 2,4,5 three levels of Cu, there is no significant difference between 1,2,3,5 four levels of Mo, so there is no significant difference between these three groups of combination matchings.
Table 7 different treatment the result of multiple comparisons
Annotate: at least contain between two processing of a same letter (capitalization or less letter) that to be illustrated on respective horizontal difference not remarkable; Do not contain between two processing of same letter (capitalization or less letter) be illustrated on respective horizontal significant difference or difference extremely remarkable.
Three, best K ion source determines
Show that by above-mentioned experiment the K ion is principal element, and after determining its optimum concn, the present invention then screens the source of K ion, confirms the impact of the potassium ion of different sources on the growth of subtilis (Bacillus subtilis) B201CGMCC No.5786.Concrete operations are as follows:
(1) take Tryptone Broth substratum is basic medium, selects respectively KH
2pO
4, K
2hPO
4, KCl, K
2sO
4, KNO
3, K
2cO
3with the source of KOH as the K ion, add the K ion that volumetric molar concentration is 2.2mM, do not add other any mineral nutriment.The Tryptone Broth substratum that does not add any mineral nutriment of take is contrast.
(2) seed liquor of subtilis (Bacillus subtilis) B201 CGMCC No.5786 is inoculated into respectively to the Tryptone Broth substratum of 7 kinds of K ions that contain different sources being prepared in step (1) with the inoculum size of volumn concentration 2%, and in the Tryptone Broth substratum that does not add any mineral nutriment in contrast, vibrate 2 minutes, add growth curve analyser (bioscreen, Finland, Oy Growth Curves Ab Ltd) in supporting culture plate, every hole adds the nutrient solution that volume is 150 μ L, every processing repeats for 3 times, the culture plate of application of sample is put into to the growth curve analyser, 32 ℃, 20h is cultivated in concussion, within every 15 minutes, survey an OD
600value, its growth conditions of on-line monitoring is drawn growth curve.
Result as shown in Figure 2.As can be seen from the figure, with K
2hPO
4for K source, the highest (OD of bacterium amount
600value is maximum), with KH
2pO
4, KCl, K
2sO
4, KOH, K
2cO
3for the K ion source, compared with the control there is to promoter action the growth of subtilis (Bacillus subtilis) B201 CGMCC No.5786, but there is no difference between these materials; And with KNO
3for the K ion source, the poor growth of subtilis (Bacillus subtilis) B201 CGMCC No.5786, although finally can reach the bacteria containing amount that other several materials are the K ion source, growth velocity is slow (the growth curve slope is less).So select with K
2hPO
4for the K ion source.
Whole results of cumulated volume embodiment, show that the proportioning of the best mineral nutriment of subtilis (Bacillus subtilis) B201 CGMCC No.5786 is: K
2hPO
40.19g/L, CuSO
45H
2o 0.002g/L is converted to the K ion and the final concentration of Cu ion in substratum is: the final concentration of K ion in Tryptone Broth substratum is 2.2mM, and the final concentration of Cu ion in Tryptone Broth substratum is 8 μ M.
The Tryptone Broth culture medium culturing genus bacillus that embodiment 2, use contain best mineral nutriment proportioning
One, in the growth curve analyser, cultivate
The Tryptone Broth substratum that contains best mineral nutriment proportioning obtained with process embodiment 1 optimization is at growth curve analyser (bioscreen, Finland, Oy Growth Curves Ab Ltd) cultivate subtilis (Bacillus subtilis) B201 CGMCC No.5786 in supporting culture plate, every hole adds the nutrient solution that volume is 150 μ L, every processing 3 repeats, the culture plate of application of sample is put into to the growth curve analyser, 32 ℃, 20h is cultivated in concussion continuously, and every 15min surveys an OD
600value.Arrange not add the Tryptone Broth substratum of any mineral nutriment in contrast simultaneously, draw growth curve.
Result as shown in Figure 3, as can be seen from the figure, subtilis (Bacillus subtilis) B201 CGMCC No.5786 grows and compares with in the Tryptone Broth substratum that does not add mineral nutriment in embodiment 1 optimizes the Tryptone Broth substratum that contains best mineral nutriment proportioning obtained, growth velocity is fast, and the bacterium amount is high.Thereby verified, the growth needs mineral nutriment of subtilis (Bacillus subtilis) B201 CGMCC No.5786, and show that the best proportioning of mineral nutriment is: K
2hPO
40.19g/L, CuSO
45H
2o 0.002g/L, the final concentration of K ion in Tryptone Broth substratum is 2.2mM, the final concentration of Cu ion in Tryptone Broth substratum is 8 μ M.
Two, in fermentor tank, cultivate
In the optimization Tryptone Broth substratum that subtilis (Bacillus subtilis) B201 CGMCC No.5786 is being added with respectively to mineral nutriment in the 50L fermentor tank and the Tryptone Broth substratum that does not add any mineral nutriment, cultivate, contrasted, observed the difference of growth velocity, bacterium output, sporulation time and the spore forming rate of bacterium.Concrete operations are as follows:
Subtilis (Bacillus subtilis) B201 CGMCC No.5786 after activation is inoculated in to the 50L seed fermentation tank that the 25L substratum is housed, initial pH value is 7.5, temperature is 32 ℃, cultivating early stage (starting to before the thalli growth logarithmic phase from cultivation) air flow is 0.5 (V/Vmin), (described air flow recently means with the volume of air by the unit volume nutrient solution in per minute 1.5 (V/Vmin) late stage of culture (finishing from the thalli growth logarithmic phase to the maximum growth amount phase), 0.5 the volume that (V/Vmin) in the expression per minute, passes into gas is half of nutrient solution volume).Within every 2 hours, get one time sample, on the one hand, detect OD
600value, until sporulation (spore forming rate reaches 80%), thereby growth curve obtained; On the other hand, micro-Microscopic observation thalli morphology, calculate spore forming rate.Wherein, utilize the microscopic examination thalli morphology to adopt Victoria Green WPB and the two methods of dying of sarranine, because gemma has wall thickness and the low characteristic of trafficability characteristic, it generally is difficult to be colored painted, but after being colored, it is difficult to again be washed off, therefore by strong staining agent Victoria Green WPB, it is dyeed, the colour contrast between thalline and gemma is just apparent in view, just makes gemma (green) and thalline (redness) make a distinction.
The growth velocity of described bacterium is embodied in thalli growth curve (OD
600) slope on, slope is larger, the growth velocity of bacterium is faster.Described bacterium output is embodied in thalli growth curve (OD
600) the corresponding OD of point (X-coordinate) sometime
600value (ordinate zou) is upper, OD
600be worth greatlyr, bacterium is higher in the output of this time point.Described sporulation (spore forming rate the reaches 80%) time is embodied in fermentor tank thalli growth curve (OD
600) time point of end, the end time point is more forward, and the time that forms gemma is just shorter.Described spore forming rate is embodied in micro-lower observed data record, spore forming rate=100% * (quantity of the quantity/thalline of gemma).
The growth curve result as shown in Figure 4, subtilis (Bacillus subtilis) B201 CGMCC No.5786 34 hours spore forming rates of growth in optimizing Tryptone Broth substratum reach 90%, and cultivate in not adding the Tryptone Broth substratum of mineral nutriment, within 40 hours, spore forming rate reaches 40%.Compared with the control, added the substratum growth velocity fast (the growth curve slope is large) of mineral nutriment, high (the same time point OD of bacterium amount
600value is large), form the gemma time short (the end time point is more forward).In addition, through microscopic examination, the spore forming rate (90%) of subtilis (Bacillus subtilis) B201 CGMCC No.5786 in optimizing Tryptone Broth substratum also will be apparently higher than contrast (not forming gemma).
As can be seen from Figure 4, the final OD value of Tryptone Broth substratum group that does not add mineral nutriment in contrast is 0.436, and the time is 40 hours; And the final OD value of optimization Tryptone Broth substratum group that has added mineral nutriment is 1.207, the time used is 34 hours.So calculate, added the optimization Tryptone Broth substratum group fermented liquid bacteria containing amount raising amount of mineral nutriment=(1.207-0.436)/0.436=1.77 doubly, the sporulation time shorten: 100%(40-34)/40=15%, so calculate that production cost will reduce by 30%.
In order to detect mineral nutriment, whether the growth promoting function of subtilis (Bacillus subtilis) B201 CGMCC No.5786 is had to the substratum dependency, the present invention has also replaced the Tryptone Broth substratum described in embodiment 1 and 2 with industrial fermention medium and has carried out further research.
The industrial fermentation substratum: bean cake powder 20g/L, corn 18g/L, fish meal 5g/L, starch 4g/L, sal epsom 0.6g/L, calcium carbonate 5g/L, surplus is water, pH7.2-7.5.The concentration of above-mentioned each material is its final concentration in the industrial fermentation substratum.
Concrete operations are as follows:
(1) detect the content of K ion in various industrial raw material with atomic absorption spectrophotometer, calculate the K ion content (in Table 8) in whole industrial fermentation culture medium solution.In above-mentioned industrial fermentation substratum, not add external source K(a), and to add respectively on this basis final concentration be 0.3g/L(b), 0.5g/L(c), 0.7g/L(d) KH
2pO
4, obtain a, b, c, tetra-kinds of culture medium prescriptions of d.
(2) subtilis (Bacillus subtilis) the B201 CGMCC No.5786 after activation is inoculated in respectively in the 300mL triangular flask of the 50mL industrial fermentation substratum that 4 kinds of K ions that contain different sources preparing in step (1) are housed, initial pH value is 7.5, temperature is 32 ℃, rotating speed is 160rpm, within 5 hours, get sample one time, micro-Microscopic observation thalli morphology, until spore forming rate (sediments microscope inspection, calculate spore forming rate, specifically referring to embodiment 2 step 2 correlation step) reach 80% and stop cultivating, detect final bacteria containing amount with counting method of blood cell.Blood counting chamber counting operation method is as follows: 1. get clean blood counting chamber, clean special cap slide is placed on to graduated position on blood counting chamber.2. the gemma rate is reached to 80% fermenation raw liquid and add the magnetic stirring apparatus 45min that vibrates for rotor, therefrom take out 10mL to the 250mL triangular flask that the 90mL sterilized water is housed, add the rotor 30min that vibrates on magnetic stirring apparatus, therefrom take out again 5mL to the 100mL triangular flask that the 45mL sterilized water is housed, add the rotor 20min that vibrates on magnetic stirring apparatus, therefrom take out 1mL to the test tube that the 9mL sterilized water is housed, 2min vibrates on the vortex oscillation device again.Successively the bacterium liquid of cultivation is carried out to suitable dilution with sterilized water, with every little lattice, have 3-5 bacterium to be advisable.3. shake up the bacterium liquid of dilution, with aseptic dropper, draw a small amount of bacterium liquid, from the edge of cover plate, drip a droplet (too much unsuitable), make bacterium liquid from the nucleonics that is advanced into platform.Must not note and make in nucleonics to have bubble while adding bacterium liquid, standing about 5min.Find square grid under low power lens after, the conversion high power lens is observed and is counted; Observing 80 little lattice, with its mean number, be multiplied by 4000000, is exactly the quantity of gemma in every milliliter of bacteria suspension.4. count (tallies of 16 * 25 specifications): number of cells (individual/mL)=80 the interior bacterial count of little lattice * bacterium liquid extension rate * 4 * 10
6/ 80
The content of K in table 8 industrial fermentation raw material and industrial fermentation substratum
Result as shown in Figure 5, as can be seen from the figure, has been added KH
2pO
4b, c, d industrial fermentation substratum in bacteria containing amount be significantly higher than the industrial fermentation substratum that a does not add K, but between these three groups (b, c and d), difference is not remarkable.This has further proved the growth importance of K ion pair subtilis (Bacillus subtilis) B201 CGMCC No.5786.Still be not enough to meet the growth of subtilis (Bacillus subtilis) B201 CGMCC No.5786 only according to the content of K ion in industrial raw material, need to add the K ion of external source, thereby promote the growth of subtilis (Bacillus subtilis) B201 CGMCC No.5786, the considering cost problem, finally determine and add the KH that final concentration is 0.3g/L on the basis of industrial fermentation culture medium prescription
2pO
4growth the best to subtilis (Bacillus subtilis) B201 CGMCC No.5786.This result with in embodiment 1 and 2, adopt Tryptone Broth substratum consistent as the result of basic medium, prove mineral nutriment to the growth promoting function of subtilis (Bacillus subtilis) B201 CGMCC No.5786 without the substratum dependency.
Subtilis involved in the present invention (Bacillus subtilis) B201 CGMCC No.5786 can be applicable to the control of cucumber fusarium axysporum, specific as follows:
The experiment place is China Agricultural University's research park greenhouse, and the time is that in April, 2008 is to May.In greenhouse, day temperature is 28 ℃ of 24 –, and evening temperature is 20 ℃ of 15 –, and humidity is 60%-80%.The variety name of cucumber seeds is the close thorn in Xintai City, purchased from close thorn seed of Fructus Cucumidis sativi field, Xintai City, Shandong Province.
Bacillus subtilis strain B068150(claims again bacterial strain B068150, is a strain biocontrol microorganisms): the public can obtain from China Agricultural University; Reference: Li Lihua, Wang Qi. screening and the evaluation [A] of control cucumber fusarium axysporum endophytic Bacillus. the 4th collection of thesis .2008 of boundary Chinese Plants Micobial Disease scientific seminar, 56..
Fusarium oxysporum cucumber specialized form (Fusarium oxysporum f.sp.cucumerinum): the public can obtain from China Agricultural University; Reference: Mandeel, Q., and Baker, R.Mechanisms involved in biological control of Fusarium of cucumber with strains of nonpathogenic Fusarium oxysporum.Phytopathology.1991.81:462-469.
The preparation of spore suspending liquid: subtilis (Bacillus subtilis) B201 CGMCC No.5786 is seeded to the extractum carnis liquid nutrient medium and (is formed by 3g extractum carnis, 10g peptone, 5g NaCl and 1000mL water, pH7.0-7.2), 30 ℃, 200rpm shaking culture 6-7 days, then centrifugal (centrifugal 5 minutes of 5000rpm), collecting precipitation (gemma), with sterilized water, the bud gemma is suspended, obtaining gemma concentration is 1 * 10
8cFU mL
-1spore suspending liquid.
Carry out following four groups of parallel processing (the nutrition pot diameter is 9cm * 9cm, each nutrition pot kind 5 strain, every group arranges three nutrition pots):
First group (experimental group 1): by the cucumber seeds surface sterilization, be placed in sterile petri dish (filter paper moisturizing, two-layer gauze covering) being cultured to seed shows money or valuables one carries unintentionally, then be transferred in the spore suspending liquid of above-mentioned preparation and soak 1 hour, then be transferred to again (filter paper moisturizing in sterile petri dish, two-layer gauze covers) surely grow and cultivate 24 hours, then (with sterilized water suspension Fusarium oxysporum cucumber specialized form, spore concentration is 5 * 10 then to be transferred to Fusarium oxysporum cucumber specialized form spore suspension
6cFU mL
-1) in soak 30 minutes, more then be transferred in sterile soil and cultivate 24 days;
Second group (experimental group 2): by the cucumber seeds surface sterilization, be placed in sterile petri dish (filter paper moisturizing, two-layer gauze covers) be cultured to seed and show money or valuables one carries unintentionally, (with the gemma of sterilized water suspension biocontrol strain B068150, gemma concentration is 1 * 10 then to be transferred to the spore suspending liquid of bacterial strain B068150
8cFU mL
-1) the middle immersion 1 hour, then be transferred to again (filter paper moisturizing in sterile petri dish, two-layer gauze covers) surely grow and cultivate 24 hours, then (with sterilized water suspension Fusarium oxysporum cucumber specialized form, spore concentration is 5 * 10 then to be transferred to Fusarium oxysporum cucumber specialized form spore suspension
6cFU mL
-1) in soak 30 minutes, more then be transferred in sterile soil and cultivate 24 days;
The 3rd group (experimental group 3): by the cucumber seeds surface sterilization, be placed in sterile petri dish (filter paper moisturizing, two-layer gauze cover) and be cultured to seed and show money or valuables one carries unintentionally, (dissolve derosal with sterilized water, the concentration of derosal is 0.01g mL then to be transferred to carbendazim solution
-1) the middle immersion 1 hour, then be transferred to again (filter paper moisturizing in sterile petri dish, two-layer gauze covers) surely grow and cultivate 24 hours, then (with sterilized water suspension Fusarium oxysporum cucumber specialized form, spore concentration is 5 * 10 then to be transferred to Fusarium oxysporum cucumber specialized form spore suspension
6cFU mL
-1) in soak 30 minutes, more then be transferred in sterile soil and cultivate 24 days;
The 4th group (control group): by the cucumber seeds surface sterilization, be placed in sterile petri dish (filter paper moisturizing, two-layer gauze covering) being cultured to seed shows money or valuables one carries unintentionally, then continue surely to grow to cultivate 24 hours, then (with sterilized water suspension Fusarium oxysporum cucumber specialized form, spore concentration is 5 * 10 to be transferred to Fusarium oxysporum cucumber specialized form spore suspension again
6cFUmL
-1) in soak 30 minutes, more then be transferred in sterile soil and cultivate 24 days.
5 grades of the Seriousness gradation standards of cucumber fusarium axysporum Seedling Stage morbidity, specific as follows: 0, the growth of plant root, stem and leaf is normal; 1,1/4th following rhizome flavescence, plant is slightly downgraded; 2,1/4th to 1/2nd rhizome flavescence, the bottom vein fades; 3,1/2nd to 3/4ths rhizome flavescence, the basal part of stem lobe; 4, the direct withered death of the rhizome flavescence more than 3/4ths, or plant.
Disease index=∑ (diseased plant numbers at different levels * this disease level value)/(investigating total strain number * superlative degree value) * 100.
Preventive effect (%)=(control group disease index-experimental group disease index)/control group disease index * 100.
The results are shown in Table 8.The preventive effect of bacillus subtilis B 2 01 has reached 58.66%, the preventive effect of bacterial strain B068150 has reached 50.03%, and the preventive effect of derosal is 59.98%, difference is not remarkable, further illustrate, bacillus subtilis B 2 01 has prevention effect preferably for cucumber fusarium axysporum, and prevention effect is better than bacterial strain B068150.
The potted plant biocontrol effect of table 8 bacillus subtilis B 2 01 (data are three mean values that repeat)
| Process | Disease index (%) (P 0.05) | Preventive effect |
| First group | 6.89b | 58.66 |
| Second group | 8.33ab | 50.03 |
| The 3rd group | 6.67b | 59.98 |
| The 4th group | 16.67a | -- |
Annotate: letter is different means that there were significant differences.
Claims (7)
1. cultivating the substratum of subtilis (Bacillus subtilis) B201CGMCC No.5786, is to add K in Tryptone Broth substratum
+to its final concentration be 2.2mM, add Cu simultaneously
2+to its final concentration substratum that is 8 μ M; Consisting of of Tryptone Broth substratum: the NaCl that the sucrose that the Tryptones that final concentration is 10g/L, final concentration are 10g/L, final concentration are 5g/L, surplus are water, pH7.5.
2. substratum according to claim 1, is characterized in that: described K
+derive from K
2hPO
4or K
2hPO
4hydrate.
3. substratum according to claim 1 and 2, is characterized in that: described Cu
2+derive from CuSO
4or CuSO
4hydrate.
4. the application of arbitrary described substratum in (1)-(4) are arbitrary as follows in claim 1-3:
(1) improve genus bacillus (Bacillus sp.) growth velocity;
(2) improve genus bacillus (Bacillus sp.) output;
(3) shorten genus bacillus (Bacillus sp.) the sporulation time;
(4) improve genus bacillus (Bacillus sp.) spore forming rate.
5. cultivate the method for genus bacillus (Bacillus sp.), comprise described genus bacillus (Bacillus sp.) is inoculated in claim 1-3 in arbitrary described substratum, 30-32 ℃ of step of carrying out shaking culture or aerated culture.
6. method according to claim 5, it is characterized in that: the time of described cultivation is 7-64h.
7. method according to claim 5, it is characterized in that: the time of described cultivation is 34-64h; Air flow in described culturing process is 0.5V/Vmin to 1.5V/Vmin.
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