CN110184304A - A kind of bacillus HN-2 compound killing pathogenetic bacteria and preparation method - Google Patents
A kind of bacillus HN-2 compound killing pathogenetic bacteria and preparation method Download PDFInfo
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Abstract
The present invention relates to biocontrol microorganisms field, a kind of bacillus HN-2 compound for killing pathogenetic bacteria and preparation method are disclosed.Such compound is to extract to obtain from Bei Laisi bacillus (Bacillus velezensis) HN-2, and described its deposit number of Bei Laisi bacillus HN-2 is CCTCC NO:M 2018382.Show that the fermentation liquid of the bacterial strain can directly obtain effective component by simple process after study, there is the efficient ability for inhibiting xanthomonas Xoc, Xoo, Xcc growth, and have preferable tolerance to strong acid and strong base, high temperature and ultraviolet light.Its bacteriostatic activity is more preferable than commonly using medicament Yekuzuo on the market, has the potential for being developed into the medicament that a kind of novel prevention and treatment Xanthomonas campestris causes bacteriosis.
Description
Technical field
The present invention relates to biocontrol microorganisms field, especially a kind of bacillus HN-2 compound for killing pathogenetic bacteria and preparation side
Method.
Background technique
Paddy bacterial disease is the important disease of one kind in China's Rice Production.By taking bacterial leaf-blight and cecospora spot as an example,
The long-term occurring area of mesh first two disease causes Rice Yield Loss Caused up to 10%-20% at 80,000,000 mu or more, reachable when serious
50% or more even has no harvest.
Chemical control is still the indispensable important means for currently preventing and treating the disease, but medicament is to the control efficiency of disease
It is all undesirable always, it is mainly manifested in that available pharmacy variety resource plaque is weary, and 1 dose of activity of medicine is low.Common prevention and treatment paddy bacterial
The medicament of disease has: the medicaments such as ambam, Sankel, 5- oxyphenazine, Yekuling, gram bacterium are strong, the withered phosphorus of rice, Bai Ye subtract.Thiophene
(Chuanhua-018) also known as, MBAMT, chemical name: N, N- methylene-bis--(2- amino -5- sulfydryl -1,3,4- thiadiazoles).The medicine
It is synthesized by Sichuan chemical industry institute within agent 1974, there is absorbability, which has protection and therapeutic effect, and the residual period is long, and toxicity is low, right
Crop is to prevent and treat the ideal medicament of rice bacterial leaf spot without phytotoxicity.It has become in rice in China bacterial leaf spot chemoprevention at present
Using the maximum kind of area.
Oppose withered azoles and oppose withered double, be the derivative of thiophene, two medicaments to the control efficiency of bacterial blight of rice compared with
It is excellent, it is the specific drug for preventing and treating bacterial blight of rice.They all have stronger absorbability, and uptake and transmission can be generated in 2h
Effect.But because of toxicity problem, there is teratogenesis to big white mouse and be prohibited from using.
Bacillus (Bacillus) is a kind of bacterium of current most study, is aerobic or amphimicrobian Gram
Positive bacillus, it is widely distributed, it is many kinds of, specifically include that bacillus subtilis (Bacillus subtilis), solution starch bud
Spore bacillus (Bacillus amyloliquefaciens), bacillus polymyxa (Bacillus polymyxa), wax-like gemma bar
Bacterium (Bacillus.ceres), bacillus megaterium (Bacillus megaterium) and bacillus licheniformis (Bacillus
Licheniformis) etc. (Cheng Liang etc., 2003).The 1950s, Gutter et al. reported one plant of bacillus subtilis for the first time
Bacterium thus opens gemma to causing a variety of fungal diseases of citrus to have very strong inhibiting effect (Gutter et al., 1953)
Situation of the bacillus as biological control.Inhibit pathogen since bacillus has, plant growth, induction is promoted to generate Genes For Plant Tolerance
The characteristics (Liu Peifu, 2011) such as property, cause the concern of lot of domestic and foreign Research Team.The Biocontrol Mechanism of bacillus, mainstream
Viewpoint there are three types of: the Competition between the generation of active material, inducing plant resistance and bacterial strain.During application,
Have found many problems, bacillus colonizes activity decline in the soil, in soil influenced that people is not achieved by factors
Desired effect, a series of problems, such as maintaining the quantity of population is difficult under wild environment.Therefore, it is living directly to extract biological bacteria
Property ingredient become one of the Hot Contents of current plant disease prevention and control research.
Summary of the invention
The present invention proposes a kind of bacillus HN-2 compound for killing pathogenetic bacteria and preparation method, using from Bei Laisi bud
Spore bacillus extracts to obtain compound, has preferable inhibitory effect to a variety of pathogenetic bacterias.
The technical scheme of the present invention is realized as follows:
A kind of bacillus HN-2 compound killing pathogenetic bacteria, such compound are from Bei Laisi bacillus HN-2
What extraction obtained, described its deposit number of Bei Laisi bacillus HN-2 is CCTCC NO:M2018382.
Further, extracted from Bei Laisi bacillus HN-2 reagent used in such compound be n-butanol, hydrochloric acid and/
Or ammonium sulfate.
A kind of preparation method for the bacillus HN-2 compound killing pathogenetic bacteria, step include:
(1) by the Biocontrol Strain HN-2 activation culture of preservation, fermentation liquid is obtained；Fermentation liquid centrifugation removal thallus, retains
Supernatant is i.e. without thallus fermentation liquid；
(2) extracting n-butyl alcohol is added without thallus fermentation liquid, takes upper layer n-butanol layer, removal n-butanol to obtain the final product.
Further, in the step (2), the volume ratio of the n-butanol of no thallus fermentation liquid and addition is 1:1.
A kind of preparation method for the bacillus HN-2 compound killing pathogenetic bacteria, step include:
(1) by the Biocontrol Strain HN-2 activation culture of preservation, fermentation liquid is obtained；Fermentation liquid centrifugation removal thallus, retains
Supernatant is up to no thallus fermentation liquid；
(2) titration with hydrochloric acid to pH value is added without thallus fermentation liquid is 2, refrigerates after sealing, is centrifuged later, discards supernatant liquid,
Obtain precipitating.
Further, in the step (2), the molar concentration for titrating hydrochloric acid used is 2mol/L；The condition of refrigeration is 4
DEG C refrigeration 12 hours, 10000 revs/min of revolving speed were controlled when centrifugation, is centrifuged 10 minutes.
A kind of preparation method for the bacillus HN-2 compound killing pathogenetic bacteria, step include:
(1) by the Biocontrol Strain HN-2 activation culture of preservation, fermentation liquid is obtained；Fermentation liquid centrifugation removal thallus, retains
Supernatant is i.e. without thallus fermentation liquid；
(2) ammonium sulfate powder is added without thallus fermentation liquid, after standing, centrifugation discards supernatant liquid, obtains precipitating.
Further, in the step (2), 4 DEG C stand 12 hours.
Further, the Biocontrol Strain HN-2 fermentation liquid the preparation method comprises the following steps: by the Biocontrol Strain HN-2 of preservation
28 DEG C of activation cultures on LB culture medium are inoculated in, are cultivated 2 days, the appropriate biocontrol microorganisms HN-2 of picking is added to the LB culture medium of liquid
In, 28 DEG C of shaking table, 180 rpms are shaken bacterium 48 hours to get fermentation liquid.
Further, fermentation liquid centrifugal condition are as follows: 10000 rpms are centrifuged 10 minutes.
The bacillus HN-2 compound obtained above for killing pathogenetic bacteria inhibits xanthomonas Xoc (rice in preparation
Xanthomonas campestris dwells rice pvs oryzae and oryzicola), Xoo (Xanthomonas Oryzae Pv. Oryzae), Xcc (Xanthomonas campestris pv campestris
Pvs oryzae and oryzicola) application on cause of disease bacteria preparation.
Beneficial effects of the present invention:
Biocontrol bacterial strain used in the present invention is one plant of Bei Laisi bacillus that separation screening obtains from cowpea
(Bacillus velezensis) HN-2 was carried out on 06 20th, 2018 in China typical culture collection center
Preservation, deposit number are CCTCC NO:M 2018382, and preservation address is in the Wuhan University of Wuhan, China.Table after study
Bright, the fermentation liquid of the bacterial strain can directly obtain effective component by simple process, have efficient inhibition xanthomonas
The ability of Xoc, Xoo, Xcc growth, and have preferable tolerance to strong acid and strong base, high temperature and ultraviolet light.Wherein by just
The active constituent fungistatic effect that the mode of butanol, before immunoassay extracts is best.And n-butanol extract has good stability, in strong acid
Property environment under activity be unaffected, can also keep 68.52% activity under strongly acidic conditions, ultraviolet light irradiate 3h and
Extracting solution still has very high bacteriostatic activity after handling 30min extract at 121 DEG C.
The present invention extracted from Bei Laisi bacillus obtain its bacteriostatic activity of compound than on the market commonly use medicament leaf it is withered
Azoles is more preferable, is not likely to produce drug resistance.Therefore have and be developed into the medicament that a kind of novel prevention and treatment Xanthomonas campestris causes bacteriosis
Potential.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention without any creative labor, may be used also for those of ordinary skill in the art
To obtain other drawings based on these drawings.
Fig. 1 is the obtained compound of three kinds of extracting modes to Xcc inhibitory effect；
Fig. 2 is the obtained compound of three kinds of extracting modes to Xoc inhibitory effect；
Fig. 3 is the obtained compound of three kinds of extracting modes to Xoo inhibitory effect；
Fig. 4 is the stability test result for the compound that extracting n-butyl alcohol obtains；
In figure, 1-6 is followed successively by compound ultraviolet light irradiation 0.5h-3h, and 7 be compound original sample, and 8-12 is followed successively by compound
40 DEG C of -121 DEG C of Temperature Treatment 30min, 13 be compound original sample, and 14-18 is followed successively by 40 DEG C of -121 DEG C of Temperature Treatments of compound
60min, 19-32 are followed successively by compound PH=1-14 processing for 24 hours, and 33 be compound original sample；
Fig. 5 is that ultraviolet irradiation time tests compound stability
Fig. 6 is that Temperature Treatment tests compound stability
Fig. 7 is that pH tests compound stability
Fig. 8 is four kinds of medicaments to Xoo inhibitory effect.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
N-butanol extraction prepare compound
The Biocontrol Strain HN-2 of preservation is inoculated in 28 DEG C of activation cultures on LB culture medium, is cultivated 2 days, picking is given birth in right amount
Fungi-proofing HN-2 is added in the LB culture medium of liquid, and 28 DEG C of shaking table, 180 rpms are shaken bacterium 48 hours to get fermentation liquid, benefit is arrived
With supercentrifuge, 10000 rpms of centrifugations, 10 minutes removal thallus retain supernatant up to no thallus fermentation liquid.
N-butanol is added in the ratio that no thallus fermentation liquid and n-butanol are 1:1, is sufficiently mixed two kinds of solution rear static
Overnight (12 hours), upper layer n-butanol is obtained by extraction with separatory funnel, using Rotary Evaporators, steams organic solvent n-butanol i.e.
Obtain compound described herein.
Embodiment 2
Hydrochloric acid precipitation method prepare compound
It is made referring to the method for embodiment 1 without thallus fermentation liquid, with the titration with hydrochloric acid of 2mol/L, adjusts fermentating liquid PH value,
Stop that hydrochloric acid is added dropwise when making pH value adjustment to 2, sealing is put into 4 DEG C of refrigerator cold-storages overnight (12 hours), recycles supercentrifuge 4
DEG C, it 10000 rpms, is centrifuged 10 minutes, discards supernatant liquid, precipitated, precipitating is compound described herein.
Embodiment 3
Ammonium sulfate is saturated precipitation method prepare compound
It is made referring to the method for embodiment 1 without thallus fermentation liquid, 10000 rpms of centrifugation 10min of bacterium solution are slowly added to
Ammonium sulfate powder, protein precipitation, 4 DEG C stand overnight (12 hours), and centrifugation is precipitated, compound as described herein.
For the ease of saving, compound obtained in above-described embodiment is dried to obtain dry powder using freeze drier
It is saved again.
The fungistatic effect of compound described herein is tested
Respectively by the ammonium sulfate precipitation of HN-2 thallus fermentation liquid, acid precipitating and the freeze-dried rear formation of n-butanol extract
Powder by concentration be that 10 μ L are added dropwise on the filter paper that diameter is 6mm after 50mg/mL wiring solution-forming and wait its drying, will
Xoc, Xoo, Xcc bacterium solution are on 9cmLB culture plate in diameter with the uniform scumbling of swab stick.It is placed in LB plate center and has been added dropwise 10
The diameter of μ l sterile water is the filter paper of 6mm.And place filter paper respectively at the three of distance center 2.5cm, culture dish is inverted
It is put into cultivate in 28 DEG C of camera bellows and carries out antibacterial observation afterwards for 24 hours, repeat three groups.
Bacteriostatic test shows: three kinds of active component extracts of biocontrol microorganisms HN-2 fermentation liquid have inhibition to imitate three kinds of bacterium
Fruit, and the active substance highest extracted with the mode of extracting n-butyl alcohol, to the antibacterial circle diameter of Xoc be 41.57 ±
0.33mm, the antibacterial circle diameter to Xoo are 38.60 ± 0.08mm, and the antibacterial circle diameter to Xcc is 28.49 ± 0.11mm.It can be with
Find out, n-butanol extract is best to the inhibitory effect of Xoc bacterium.(data are shown in Table 1-3 and Fig. 1-3)
1 three kinds of extracting modes of table are to Xoo inhibitory effect data
2 three kinds of extracting modes of table are to Xoc inhibitory effect data
3 three kinds of extracting modes of table are to Xcc inhibitory effect data
N-butanol extract tests the median effective concentration of xanthomonas Xoc, Xoo, Xcc
Xanthomonas Xoc, Xoo, Xcc bacterium of preservation is inoculated on appropriate media to (Xoc, Xoo bacterium is suitable for respectively
Culture medium is PSA culture medium, and the appropriate media of Xcc bacterium is NYG culture medium) 28 DEG C of activation cultures, it cultivates 2 days, appropriate picking
Xanthomonas Xoc, Xoo, Xcc bacterium is added separately in the appropriate media of liquid, and 28 DEG C of shaking table, 180 rpms are shaken bacterium
After for 24 hours, takes 400 μ L bacterium solutions to be added in advance plus in the 15mL centrifuge tube of good 4.5mL appropriate liquid culture medium, mix.Take 1 respectively again
μ L, 2 μ L, 5 μ L, 10 μ L, 25 μ L, 50 μ L, 100 μ L, 200 μ L concentration are molten for the HN-2 fermentation liquid n-butanol extract of 50mg/mL
Liquid in 15mL centrifuge tube (i.e. final concentration be respectively as follows: 10 μ g/mL, 20 μ g/mL, 50 μ g/mL, 100 μ g/mL, 250 μ g/mL and
500 μ g/mL, 1mg/ μ L and 2mg/ μ L), it mixes and (passes through test of many times when surveying HN-2 n-butanol extract to Xoo MIC50 value
Added drug concentration is different when finding most suitable drug concentration, therefore testing from other two bacterium, it is seen that table 4).It is put into 28 DEG C, 180 turns
It is cultivated in shaking table per minute and measures spectrophotometric value afterwards for 24 hours, MIC50 value is calculated by SPSS software.By calculating, HN-2 bacterium
Strain fermentation liquid to Xoo bacterium MIC50 value close to 0 μ g/mL,.It is 4.605 μ g/mL to Xoc bacterium MIC50 value, 95% confidence interval is
0.171-16.791μg/mL.It is 36.730 μ g/mL to Xcc bacterium MIC50 value, 95% confidence interval is 1.289-118.773 μ g/
mL.(data are shown in Table 4-6).
4 HN-2 bacterial strain fermentation liquor of table records Xoo MIC50
5 HN-2 bacterial strain fermentation liquor of table records Xoc MIC50
6 HN-2 bacterial strain fermentation liquor of table records Xcc MIC50
Biocontrol microorganisms HN-2 fermentation liquid n-butanol extract stability test
(1) UV stable
N-butanol extract (5mg/mL) is placed in the culture dish of sterilizing.In ultraviolet lamp (under aseptic operating platform) respectively according to
Penetrate 30,60,90,120,150, after 180min, measure activity change with filter paper enzyme, the bacterium used is xanthomonas Xoo.It is right
According to the n-butanol extract to be irradiated without ultraviolet light.It is found by experiment that the extract solution molten after ultraviolet light irradiates 3h
Liquid antibacterial circle diameter is 27.2 ± 0.24mm, compared to 29.80 ± 0.34mm of antibacterial circle diameter of control group, it can be seen that is extracted
Object still has very high bacteriostatic activity, and active constituent is through stablizing (data are shown in Table 7 and Fig. 4 and Fig. 5) under ultraviolet irradiation.
(2) temperature stability
N-butanol extract (5mg/mL) is placed at 40,60,80,100 and 121 DEG C and handles 30min and 60min respectively.
Activity change is measured with filter paper enzyme after cooling, the bacterium used is xanthomonas Xoo.Control is extracted for n-butanol at room temperature
Object.After being found by experiment that the extract solution handles 30min under the conditions of 100 DEG C, antibacterial circle diameter size be 28.79 ±
0.49mm is of little difference compared with 28.40 ± 0.23mm of antibacterial circle diameter size of control group.30min is handled at 121 DEG C
Afterwards, inhibition zone size is 24.70 ± 0.53mm, and bacteriostatic activity is the 86.91% of original solution, activity still with higher
(data are shown in Table 8 and table 9 and Fig. 4 and Fig. 6).
(3) pH stability
The pH value of n-butanol extract (5mg/mL) is adjusted to 1 respectively with 2.5mol/L NaOH and 6mol/L HCl, 2,3,
4,5,6,7,8,9,10,11,12,13,14, it stands and measures activity change with filter paper enzyme after pH is recalled to 7 afterwards for 24 hours, use
Bacterium is xanthomonas Xoo.Control is the n-butanol extract solution dissolved with sterile water.It is found by experiment that the extract is molten
After liquid is handled for 24 hours under pH≤9, antibacterial circle diameter size is 24.25-26.19mm, big compared to control group antibacterial circle diameter
Small 28.94 ± 0.56mm, bacteriostatic activity still keep higher level.The extract solution is handled for 24 hours under 9 alkaline condition of pH >
Afterwards, antibacterial circle diameter size is 19.83-22.88mm, wherein bacteriostatic activity is after handling under pH=14 basic conditions
68.52% (data are shown in Table 10 and Fig. 4 and Fig. 7) of original solution.
Impact effect of 7 ultraviolet light of table to HN-2 bacterial strain fermentation liquor
Impact effect of the 8 Temperature Treatment 30min of table to HN-2 bacterial strain fermentation liquor
Impact effect of the 9 Temperature Treatment 60min of table to HN-2 bacterial strain fermentation liquor
Influence of 10 ultraviolet light of table to HN-2 bacterial strain fermentation liquor
N-butanol extract and Yekuzuo, bacitracin, kasugarnycin expression activitiy are tested
HN-2 n-butanol extract, 97% Yekuzuo raw medicine, bacitracin and 6% kasugarnycin aqua are made into 10mg/
The solution of mL concentration, which has, to be added dropwise 10 μ L on the filter paper that diameter is 6mm and waits its drying, is added dropwise using sterile water as control
On another filter paper.By Xoo bacterium solution with the uniform scumbling of swab stick after the LB culture plate that diameter is 9cm, cultivated in distance
Filter paper is placed respectively at the 2.5cm of ware center, is sealed.Culture dish is inverted into cultivate in 28 DEG C of camera bellows carries out antibacterial sight afterwards for 24 hours
It examines, repeats three groups.
Bacteriostatic test shows: four kinds of medicaments have bacteriostatic activity to Xoo bacterium, wherein biocontrol microorganisms HN-2 n-butanol extract
Bacteriostatic activity is best, is secondly bacitracin.Biocontrol microorganisms HN-2 n-butanol extract to Xoo antibacterial circle diameter be 29.79 ±
0.18mm；Bacitracin is 27.71 ± 0.64mm to Xoo antibacterial circle diameter size；Kasugarnycin is to Xoo antibacterial circle diameter size
18.71±0.10mm；Yekuzuo is 16.35 ± 0.45mm to Xoo antibacterial circle diameter size.(data are shown in Table 11 and Fig. 8).
11 4 kinds of medicaments of table are to Xoo fungistatic effect data
In summary test result, it can be seen that biocontrol microorganisms HN-2 have it is efficient inhibit xanthomonas Xoc, Xoo,
The ability of Xcc growth, wherein the active constituent fungistatic effect extracted by way of extracting n-butyl alcohol is best.And by just
The active constituent that the mode of butanol, before immunoassay extracts has preferable tolerance to strong acid and strong base, high temperature and ultraviolet light, in strong acid
Property environment under activity be unaffected, can also keep 68.52% activity under strongly acidic conditions, ultraviolet light irradiate 3h and
Extracting solution still has very high bacteriostatic activity after handling 30min extract at 121 DEG C.Than commonly using medicament Yekuzuo, spring on the market
The bacteriostatic activity of thunder mycin and bacitracin is more preferable, and compound described herein is not likely to produce drug resistance.Therefore, the application
The compound has the potential for being developed into the medicament that a kind of novel prevention and treatment Xanthomonas campestris causes bacteriosis.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of bacillus HN-2 compound for killing pathogenetic bacteria, it is characterised in that: such compound is from Bei Laisi gemma
Extraction obtains in bacillus HN-2, and described its deposit number of Bei Laisi bacillus HN-2 is CCTCC NO:M2018382.
2. a kind of bacillus HN-2 compound for killing pathogenetic bacteria as described in claim 1, it is characterised in that: from Bei Laisi
It is n-butanol, hydrochloric acid and/or ammonium sulfate that reagent used in compound is extracted in bacillus HN-2.
3. a kind of preparation method for the bacillus HN-2 compound for killing pathogenetic bacteria as claimed in claim 1 or 2, feature
It is, step includes:
(1) by the Biocontrol Strain HN-2 activation culture of preservation, fermentation liquid is obtained；Fermentation liquid centrifugation removal thallus, retains supernatant
Liquid is i.e. without thallus fermentation liquid；
(2) extracting n-butyl alcohol is added without thallus fermentation liquid, takes upper layer n-butanol layer, removal n-butanol to obtain the final product.
4. a kind of preparation method for the bacillus HN-2 compound for killing pathogenetic bacteria as claimed in claim 3, feature exist
In: in the step (2), the volume ratio of the n-butanol of no thallus fermentation liquid and addition is 1:1.
5. a kind of preparation method for the bacillus HN-2 compound for killing pathogenetic bacteria as claimed in claim 1 or 2, feature
It is, step includes:
(1) by the Biocontrol Strain HN-2 activation culture of preservation, fermentation liquid is obtained；Fermentation liquid centrifugation removal thallus, retains supernatant
Liquid is up to no thallus fermentation liquid；
(2) titration with hydrochloric acid to pH value is added without thallus fermentation liquid is 2, refrigerates after sealing, is centrifuged later, discard supernatant liquid, obtains
Precipitating.
6. a kind of preparation method for the bacillus HN-2 compound for killing pathogenetic bacteria as claimed in claim 5, feature exist
In: in the step (2), the molar concentration for titrating hydrochloric acid used is 2mol/L；The condition of refrigeration is that 4 DEG C of refrigerations 12 are small
When, 10000 revs/min of revolving speed are controlled when centrifugation, are centrifuged 10 minutes.
7. a kind of preparation method for the bacillus HN-2 compound for killing pathogenetic bacteria as claimed in claim 1 or 2, feature
It is, step includes:
(1) by the Biocontrol Strain HN-2 activation culture of preservation, fermentation liquid is obtained；Fermentation liquid centrifugation removal thallus, retains supernatant
Liquid is i.e. without thallus fermentation liquid；
(2) ammonium sulfate powder is added without thallus fermentation liquid, after standing, centrifugation discards supernatant liquid, obtains precipitating.
8. a kind of preparation method for the bacillus HN-2 compound for killing pathogenetic bacteria as claimed in claim 7, feature exist
In: in the step (2), 4 DEG C stand 12 hours.
9. a kind of preparation method for the bacillus HN-2 compound for killing pathogenetic bacteria as claimed in claim 1 or 2, feature
Be: the Biocontrol Strain HN-2 fermentation liquid is trained the preparation method comprises the following steps: the Biocontrol Strain HN-2 of preservation is inoculated in LB
28 DEG C of activation cultures on base are supported, are cultivated 2 days, the appropriate biocontrol microorganisms HN-2 of picking is added in the LB culture medium of liquid, and 28 DEG C of shaking table,
180 rpms are shaken bacterium 48 hours to get fermentation liquid.
10. a kind of preparation method for the bacillus HN-2 compound for killing pathogenetic bacteria as claimed in claim 9, feature exist
In: fermentation liquid centrifugal condition are as follows: 10000 rpms are centrifuged 10 minutes.
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| CN110117556A (en) * | 2019-03-27 | 2019-08-13 | 海南大学 | A kind of efficient biocontrol microorganisms HN-2 and zymotechnique for producing lipopeptid substance |
| CN113785842A (en) * | 2021-10-20 | 2021-12-14 | 海南大学 | Pesticide composition for preventing and treating plant fungal diseases and preparation method thereof |
| CN115413676A (en) * | 2022-07-24 | 2022-12-02 | 海南省农业科学院热带果树研究所 | Microbial preparation with growth promoting effect |
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| CN110117557A (en) * | 2019-03-27 | 2019-08-13 | 海南大学 | A kind of biocontrol microorganisms HN-2 and extract inhibit the application on disease fungus preparation in preparation |
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| CN115413676A (en) * | 2022-07-24 | 2022-12-02 | 海南省农业科学院热带果树研究所 | Microbial preparation with growth promoting effect |
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