CN104673716A - Sfp gene-deleted biocontrol bacteria as well as fermentation technology and application thereof - Google Patents

Sfp gene-deleted biocontrol bacteria as well as fermentation technology and application thereof Download PDF

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CN104673716A
CN104673716A CN201510061973.0A CN201510061973A CN104673716A CN 104673716 A CN104673716 A CN 104673716A CN 201510061973 A CN201510061973 A CN 201510061973A CN 104673716 A CN104673716 A CN 104673716A
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biocontrol microorganisms
fermented liquid
hab
antibacterial
fermented
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CN104673716B (en
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缪卫国
靳鹏飞
刘文波
郑服丛
王皓楠
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Hainan University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/10—Animals; Substances produced thereby or obtained therefrom
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Abstract

The invention provides bacillus amyloliquefaciens HAB-2, which has the collection number of CCTCC M 2015070. Although the key lipopeptide synthetase gene sfp gene is deleted, the surfactin substances can still be generated, indicating that the strain has a specific and distinctive biosynthetic pathway and has a remarkably high research value. The bacillus amyloliquefaciens HAB-2 has very strong ability of inhibiting pathogenic fungi, has a wide antibacterial spectrum and can inhibit multiple kinds of pathogenic fungi while the antibacterial effect is good and the antibacterial rate is high. Further research finds that the active ingredient can be directly obtained through simple treatment on the fermentation broth of the strain, and the antibacterial active ingredient is stable and has very high activity in strong acid and strong alkaline environments; the antibacterial activity is not changed after 24 hours of ultraviolet irradiation, and high temperature does not influence the antibacterial active ingredient; and the fermentation broth of the biocontrol bacteria realizes certain growth promoting effect on a plant.

Description

A kind of lack sfp gene biocontrol microorganisms and zymotechnique and application
Technical field
The invention belongs to microorganism field, be specifically related to a kind of lack sfp gene biocontrol microorganisms and zymotechnique and application.
Background technology
Genus bacillus is the gram positive bacterial strain that a class produces gemma, it widely distributed, physiological characteristic is enriched, be easy to be separated and cultivate, it is the important microorganism species of occurring in nature, genus bacillus can produce multiple antibacterial substance, comprises the non-ribosomal peptide antibiotics synthesized by non-ribosomal approach and the bacteriocin synthesized by rrna approach, enzyme and other activated protein class antimicrobial substances etc.
168 bacterial strains are type strains of genus bacillus, and be also the bacterial strain the most thorough of research at present, his full-length genome has checked order in 1997.With wild genus bacillus unlike type strain 168 due to the long term subculture in laboratory, there occurs the feature that thus a lot of spontaneous mutations loses a lot of wild-type, the such as spontaneous mutation of sfp gene, make 168 bacterial strains lose the ability producing lipopeptide compound, thus lack antibacterial ability.4 '-Phosphopantetheinyl transferase of sfp genes encoding is the key gene of genus bacillus non-ribosomal approach synthetic fat peptides.
The aimed strain HAB-2 of this research shows through a series of previous experiments: this bacterial strain does not have sfp gene, but we detect in HAB-2 fermented liquid again that obtain due to sfp disappearance can not getable fat peptide matters---Surfactin class material by modern instrumental analysis means, this just illustrates that the biocontrol microorganisms HAB-2 of this research has the pathways metabolism of special metabolism lipopeptid class, is worth further further investigation.
Summary of the invention
An object of the present invention is to provide a kind of biocontrol microorganisms, its disappearance sfp gene, but there is the ability producing lipopeptide compound, there is high researching value.Another object of the present invention is to provide the zymotechnique of this biocontrol microorganisms.Another object of the present invention is to provide the extracting method of the fermented product activeconstituents of this biocontrol microorganisms, and last object of the present invention is to provide the application of this biocontrol microorganisms.
First aspect of the present invention is to provide a kind of biocontrol microorganisms lacking sfp gene, for bacillus amyloliquefaciens ( bacillus amyloliquefaciens) HAB-2, deposit number is: CCTCC M 2015070.
The present invention second aspect is to provide the zymotechnique of the biocontrol microorganisms described in the present invention first aspect, biocontrol microorganisms described in the present invention first aspect is inoculated in the bacteriolyze broth culture of liquid, under the condition of 26 ~ 30 DEG C, 50 ~ 500 revs/min are shaken bacterium 1 ~ 3 day, namely obtain fermented liquid.
Preferably, also pre-treatment step is comprised: first the biocontrol microorganisms described in the present invention first aspect to be inoculated on bacteriolyze broth culture at 26 ~ 30 DEG C activation culture 1 ~ 6 day.
3rd aspect of the present invention is to provide the extracting method of the fermented product activeconstituents of the biocontrol microorganisms described in the present invention first aspect, comprises the following steps:
Step 1, adopt the zymotechnique described in the present invention second aspect to obtain fermented liquid, then centrifugal removing thalline, obtains without fermented liquid;
Step 2, without fermented liquid n-butanol extraction, gets extraction liquid, and concentrated removing propyl carbinol, to obtain final product.
4th aspect of the present invention is to provide the extracting method of the fermented product activeconstituents of the biocontrol microorganisms described in another kind of the present invention first aspect, comprises the following steps:
Comprise the following steps:
A, adopt the zymotechnique described in the present invention second aspect to obtain fermented liquid, then centrifugal removing thalline, obtains without fermented liquid;
B, without fermented liquid salt acid for adjusting pH to 1.5 ~ 3, leaves standstill at least 3h at 1 ~ 10 DEG C;
C, centrifugal, abandoning supernatant, gets precipitation, to obtain final product.
5th aspect of the present invention is to provide the extracting method of the fermented product activeconstituents of the biocontrol microorganisms described in another the present invention first aspect, comprise the following steps: 1) adopt the zymotechnique described in claim 2 ~ 3 any one to obtain fermented liquid, then centrifugal removing thalline, obtains without fermented liquid;
2) slowly add saturated ammonium sulphate, protein precipitation, at 1 ~ 10 DEG C, leave standstill at least 3h;
3) to be centrifugally precipitated, drying to be precipitated use afterwards 1/5 ~ 1/20 original fermented solution volume, pH value be 6 ~ 8 Tris-HCl dissolution precipitation, to obtain final product.
The biocontrol microorganisms that 6th aspect of the present invention is to provide described in the present invention first aspect is preparing the application in lipopeptid class inhibiting-bacteria preparation.
7th aspect of the present invention is to provide the application of biocontrol microorganisms in preparation control fungal diseases of plants preparation described in the present invention first aspect.
Wherein, described fungal diseases of plants be early blight of tomato, watermelon blight, banana blight, cotton wilt, rubber anthrax, rubber tree rod spore mould defoliation, tea zonate spot, mango anthracnose and or rubber brown root rot in one or more.
Bacterial strain provided by the invention, through multinomial taxonomic identification, is defined as a novel species of bacillus amyloliquefaciens, temporary called after bacillus amyloliquefaciens HAB-2( bacillus amyloliquefaciens. HAB-2), carried out culture presevation on January 27th, 2015 in China typical culture collection center (CCTCC), and proved survival, its preservation registration number is: CCTCC M 2015070, and address is in Wuhan University.
The lipopeptid class synthase gene sfp gene of bacillus amyloliquefaciens HAB-2 delete critical provided by the invention, it is Surfactin class material that the method combined with bacteriostatic activity according to modern separation technology determines the main active ingredient that this bacterial strain produces, this just illustrates that this bacterial strain has special distinguished biosynthetic pathway, has high researching value.Bacillus amyloliquefaciens HAB-2 provided by the invention has the ability of very strong suppression pathogenic fungi, and antimicrobial spectrum is extensive, and can suppress multiple pathogenic fungi, good antimicrobial effect, bacteriostasis rate is high, and this has no report in research in the past.Further research finds that the fermented liquid of this bacterial strain directly can obtain effective constituent through simple process, and effective bacteriostatic ingredients is stablized, very strong activity can be all had under strongly-acid and strong basicity environment, activity can not be affected, can not change at uviolizing 24 hours bacteriostatic activities, high temperature does not still affect effective bacteriostatic ingredients, and the growth of the fermented liquid of this biocontrol microorganisms to plant has the effect of certain growth promoting effects.
Accompanying drawing explanation
The specific PCR detected result of the functional gene of Fig. 1 biocontrol microorganisms of the present invention, wherein, 1:HAB-2 bacterial strain, A:: bacillus subtilis dientification of bacteria sequence (Bsu-man) 1287bp, B: Bacillus licheniformis qualification sequence (Bli-man) 1278bp, C: bacillus amyloliquefaciens qualification sequence (Bam-man) 1275bp, M:2000bp Marker.
Fig. 2 is the colonial morphology of biocontrol microorganisms of the present invention on different culture media, wherein, and A:AYBA substratum, B:BPY substratum, C:LB substratum, D:NA substratum, E: basic medium, F: fermention medium.
Fig. 3 is bacterial strain HAB-2 and 168 bacterial strain microbiotic synthesis related gene PCR primer agarose gel electrophoresis figure, wherein, and A:ituC, B:srfAB, C:fenD, D:ituB, M:2000DNA marker.
Fig. 4 is the PCR primer agarose gel electrophoresis figure of the crucial synthase gene of bacterial strain HAB-2 and genus bacillus type strain 168 lipopeptid class, wherein, M:2000Marker, A:HAB-2-ituA gene, B:B.S168-ituA gene, C:HAB-2-ituD gene, D:B.S168-ituD gene, E:HAB-2-lpa14 gene, F:B.S168-lpa14 gene, G:HAB-2-sfp gene, H:B.S168-sfp gene, I:HAB-2-mycB gene, J:B.S 168-mycB gene, K:HAB-2-fenB gene, L:B.S168-fenB gene.
Fig. 5 is bacterial strain HAB-2 and B.S168 bacterial strain bacteriostatic test result.
Fig. 6 is the bacteriostatic activity detected result of the antibacterial substance of HAB-2, and wherein, A is that gel survey slip-knot fruit crossed by propyl carbinol: 1 propyl carbinol (23.08mm), and 2 propyl carbinol gels tests have active No. 2 (12.12mm), and 3 gels tests have activity No. 3 (15.78mm); B is that post survey slip-knot fruit taken out by propyl carbinol: 4 take out post has active methyl alcohol to rush post, and 5 take out post chloroform methanol 1: 1; C is that post survey slip-knot fruit taken out by propyl carbinol: take out other cut non-activities of post; D is that slip-knot fruit surveyed by lipopeptid gellike post: 6 lipopeptid classes (14.77mm), and 7 lipopeptid classes have active No. 2 (11.09mm).
Fig. 7 is the TCL detected result of the antibacterial substance of HAB-2, and frame part is activated part, wherein, and 1 propyl carbinol, 2 propyl carbinol gel tests have active No. 2, and 3 gel tests have active No. 3, and 4 take out post has active methyl alcohol to rush post, 5 take out post chloroform methanol 1: 1,6 lipopeptid class, and 7 lipopeptid classes have active No. 2.
Fig. 8 is the photo of the bacteriostatic test detected result to Activities of Some Plants pathogenic fungi of biocontrol microorganisms of the present invention, wherein, and A: rubber anthrax ( colletotrichum gloeosporioides), B: No. 1, mango anthracnose ( colletotrichum gloeosporioides Penz), C: the mould defoliation of rubber tree rod spore ( corynespora cassiicola), D: banana blight No. 1 microspecies ( fusarium oxysporumf.sp.cubense, FOC).
Fig. 9 is the Detection of Stability result figure of the effective constituent that biocontrol microorganisms of the present invention produces, and wherein A is the fermented liquid of 25 DEG C: 2 n-butanol extracts, 4 lipopeptid class throw outs, 5 ammonium sulfate precipitation things; B is the fermented liquid of 28 DEG C: 10 n-butanol extracts, 12 lipopeptid class throw outs, 15 ammonium sulfate precipitation things.
Figure 10 is the pH of the effective constituent that the biocontrol microorganisms of invention produces, UV stable detected result, wherein, 1 is n-butanol extract former state, 2-12 is respectively n-butanol extract pH=2-12, 36 is lipopeptid class throw out former state, 13-23 is respectively lipopeptid class throw out PH=2-12, 25 is ammonium sulfate precipitation thing former state, 26-36 is respectively ammonium sulfate precipitation thing PH=2-12, 42 is propyl carbinol former state, 37-41 is respectively uv irradiating 3, 6, 9, 12, 24h, 43 lipopeptid former states, 44-48 is respectively uv irradiating 3, 6, 9, 12, 24h, 49 is ammonium sulfate precipitation former state, 50-54 is respectively uv irradiating 3, 6, 9, 12, 24h.
Embodiment
With reference to the accompanying drawings, the present invention is described further in conjunction with specific embodiments, to understand the present invention better.
one, strain identification and morphological feature
Biocontrol strain used in the present invention separation screening from Cotton rhizosphere soil obtains.Special primer is designed targetedly by known genome sequence, utilize the method that cross primer confirms mutually, according to the mutual confirmation that special primer and routine morphological are identified, progressively identify concrete kind from bacillus, find that biocontrol microorganisms of the present invention is bacillus amyloliquefaciens HAB-2 through research, its PCR detected result as shown in Figure 1.
The colonial morphology of bacillus amyloliquefaciens HAB-2 of the present invention as shown in Figure 2, wherein,
A: the colonial morphology of bacillus amyloliquefaciens HAB-2 is irregular on AYDA substratum, bacterium colony is large and smooth, and easily from picking substratum, bacterium colony is dry, in light brown;
B: bacillus amyloliquefaciens HAB-2 bacterium colony is rounded on BPY substratum, is with fold outside bacterium colony, easily from picking substratum, bacterium colony is dry, in light brown;
C: the bacillus amyloliquefaciens HAB-2 colonial morphology cultivated in LB cultivation is irregular, and bacterium colony is little, is with little fold outward, easily from picking substratum, bacterium colony is dry, is creamy white;
D: the bacillus amyloliquefaciens HAB-2 cultivated on NA substratum and three othernesses are comparatively large above, bacterium colony is irregular, and bacterium colony is moistening, in oily, is beige;
E: irregular, drier at the bacillus amyloliquefaciens HAB-2 bacterium colony of basic medium, in beige;
F: bacillus amyloliquefaciens HAB-2 bacterium colony is very little, irregular on fermention medium, and easily from picking substratum, bacterium colony is drier, in white.
two, the sequential analysis of lipopeptid class synthesis key gene
Surfactin family metabolic process key enzyme sfp gene (subtilis phosphopantetheine transferring enzyme), lacking this enzyme then may cause genus bacillus can not produce Surfactin class material, by Whole genome analysis, type strain B.S168 finds that this bacterial strain is only that imperfect this bacterial strain that just causes of sfp gene loses bacteriostasis.Show that this bacterial strain HAB-2 does not have sfp gene by experiment, but this experiment is again by modern POP technical flying time mass spectrum, the composition of test fat peptide extract, find that there is the existence of Surfactin class material, this just illustrates that this bacterial strain has the pathways metabolism of new distinguished metabolism Surfactin class material.
The sequential detection of 2.1HAB-2 and genus bacillus type strain 168 lipopeptid genoid
Experiment is picked out 4 and (is represented iturin Iturin respectively to representational lipopeptid genoid, Surfactin Surfactin, Feng Yuansu Fengycin) sequence carries out PCR, result as shown in Figure 3, show: srfAB, fenD, ituB tri-genes can be obtained by pcr amplification from HAB-2 bacterial strain, but 168 bacterial strains amplifications only can be increased and obtained fenD.
the sequential detection of 2.2 HAB-2 and the crucial synthase gene of genus bacillus type strain 168 lipopeptid class
Experiment is picked out 6 and (is represented iturin Iturin respectively to the crucial synthase gene gene in representational lipopeptid class building-up process, Surfactin Surfactin, Feng Yuansu Fengycin) sequence carries out PCR, as shown in Figure 4, result shows experimental result: HAB-2 bacterial strain can obtain key gene by PCR: ituA gene, lpa14 gene, mycB gene, fenB gene.Type strain B.S168 bacterial strain can obtain by PCR: sfp gene.
2.3 HAB-2 and B.S168 bacterial strain bacteriostatic tests
Mango anthracnose pathogenic fungi is inoculated on the PDA culture plate of 9cm.Apart from the position equidistant inoculation HAB-2 of central 2.5 cm and B.S168 bacterial strain, culture dish is inverted, be put in light culture at 28 DEG C to whole culture dish 2/3 time, carry out observing antibacterial situation.Result as shown in Figure 5.The imperfect B.S168 of causing bacterial strain due to sfp gene can not produce fat peptide matters and so there is no bacteriostatic activity, and this experimental strain HAB-2 lacks this gene but still has bacteriostatic activity.
the determination of 2.4 thin-layer chromatographies and activeconstituents
Filter paper side method alive: Colletotrichum gloeosporioides Penz in Mango cake is seeded in 100ml PDA liquid nutrient medium, 28 DEG C, 180 rpm cultivate 3-5d, filter out mycelia with 4 layers of sterile gauze and just can obtain mango anthrax spores suspension, by PDA solid medium heating for dissolving, when substratum is cooled to about 45 DEG C, in substratum: spore suspension=6: mango anthrax spores suspension joins in PDA substratum by the ratio of 1, limit edged mixing, be down flat plate and be mango anthrax spores suspension flat board, sample thief 15 μ L, after dripping the sample finish-drying on the first-class filter paper of sterilizing filter paper of 6mm, be attached on spore suspension flat board uniformly, be used for detecting the bacteriostatic activity of antibacterial substance of HAB-2.Result as shown in Figure 6.
By the method that TLC thin-layer chromatography and antipathogenic composition contrast mutually, we successfully have found activated part and some relevant characteristics in HAB-2 bacterial strain fermentation liquor, determine that activated part is fat peptide matters, and this kind of material polarity is comparatively large, result as shown in Figure 7.
2.5 flight time mass spectrum
Flight time mass spectrum detection is carried out to HAB-2 n-butanol extract, the result obtaining molecular weight according to flight time mass spectrum test is compared, and we are known and can have by output lipopeptid row material: C13-IturinA, C15-IturinB, C14-Mycosubtilin, C14-BacillomycinD, C15-BacillomycinD, C16-BacillomycinD, C15-SurfactinA, C14-SurfactinC, C-16FengycinA, C17-FengycinA, C16-FengycinB, C17-FengycinB.The result of test proves to detect Surfactin lipoid peptide material in HAB-2 n-butanol extract.
three, extraction process of active component
N-butanol extraction:
The Biocontrol Strain A178 of preservation is inoculated in 28 DEG C of activation culture on bacteriolyze broth culture, cultivate 3 days, the biocontrol microorganisms A178 choosing de-etiolation beans size joins in the bacteriolyze broth culture of liquid, 28 DEG C, 200 rpms are shaken bacterium 2 days, namely fermented liquid is obtained, utilize supercentrifuge, within centrifugal 10 minutes, remove thalline for 10000 rpms, obtain without thalline fermented liquid, propyl carbinol is added according to the ratio of 1:1 fermented liquid and propyl carbinol, that two kinds of solution fully mix, static 12 hours, separating funnel extraction obtains upper strata propyl carbinol, utilize Rotary Evaporators, steam organic solvent propyl carbinol and namely obtain effective constituent.
Acid precipitation method (the lipopeptid class precipitator method):
Obtain without thalline fermented liquid with n-butanol extraction, with the HCI of 6 moles often liter in this fermented liquid, regulate pH value, make pH value be adjusted to 2 stoppings and dripping hydrochloric acid, sealing is put into 4 DEG C of refrigerator cold-storages and is spent the night (12 hours), recycling supercentrifuge 4 DEG C, 11000 rpms, centrifugal 10 minutes, abandoning supernatant, be precipitated, namely precipitate after testing is lipopeptide compound (effective constituent).
The saturated precipitator method of ammonium sulfate:
Obtain, without thalline fermented liquid, slowly adding saturated ammonium sulphate, protein precipitation with n-butanol extraction, 4 DEG C of hold over night, centrifugally to be precipitated, drying to be precipitated afterwards with 1/10 original fermented solution volume, pH value be 6.8 Tris-HCl dissolution precipitation, namely obtain crude protein.
four, bacteriostatic test
Adopt plate face-off method on PDA culture medium flat plate, detect HAB-2 thalline antimicrobial spectrum, find that it is inhibited to the growth of all test plant pathogenic fungies.PDA substratum records antimicrobial spectrum and rejection ability result as shown in table 1 and Fig. 8 by plate diffusion process.Biocontrol microorganisms HAB-2 to rubber brown root rot ( phellinus noxius(Corner) G.H.Cunn.) bacteriostasis rate is best, bacteriostasis rate reaches 66.91%, to Pestallozzia theae ( pestalotiopsis guepinii) fungistatic effect is the poorest, bacteriostasis rate is 38.76%.Can 50-60% be reached to tropical plants pathogenic bacteria bacteriostasis rate, have good fungistatic effect.
Table 1 HAB-2 thalline antimicrobial spectrum and rejection ability
For examination pathogenic fungi Viable bacteria bacteriostasis rate
Early blight of tomato ( Alternaria solani（E.et M.）Jones et Grout.)） 60.67%
Watermelon blight ( Fusarium oxysporum f.sp.cubense,FOC） 60.77%
Banana blight ( Fusarium oxysporum f.sp.cubense,FOC） 56.08%
Cotton wilt ( Fusarium oxysporum f.sp.cubense,FOC） 62.31%
Rubber anthrax ( Colletotrichum gloeosporioides） 53.51%
The mould defoliation of rubber tree rod spore ( Corynespora cassiicola） 62.22%
Banana blight microspecies ( Fusarium oxysporum f.sp.cubense,FOC） 40.88%
Pestallozzia theae ( Pestalotiopsis guepinii） 38.76%
No. 1, mango anthracnose ( Colletotrichum gloeosporioides Penz） 49.57%
Banana blight No. 4 microspecies ( Fusarium oxysporum f.sp.cubense,FOC） 58.69%
Rubber brown root rot ( Phellinus noxius (Corner) G.H.Cunn.） 66.91%
No. 2, mango anthracnose ( Colletotrichum gloeosporioides） 61.68%
five, effective bacteriostatic ingredients stability test
Use the fermented liquid Different Extraction Method extracting method Test extraction thing stability of 7 days of 25 and 28 DEG C respectively, result as shown in Figure 9.
Get propyl carbinol and each 100 microlitres of fat peptide extract, 900 microlitre ddH2O, then regulate pH value, are 1.0 from PH from 2.0-12.0(gradient), room temperature places 24H, and repeat 3 times, test bacteriostatic activity with disc diffusion method, result is as shown in Figure 10.
UV stable: get propyl carbinol and each 100 microlitres of fat peptide extract, 900 microlitre ddH2O, are placed in the culture dish of sterilizing.Under the ultraviolet lamp of aseptic operating platform, direct irradiation 3,6,9,12,24H, in triplicate, tests bacteriostatic activity with disc diffusion method, and result as shown in Figure 10.
From Fig. 9 and 10, the fermented liquid of bacterial strain of the present invention directly can obtain effective constituent through simple process, and effective bacteriostatic ingredients is stablized, very strong activity can be all had under strongly-acid and strong basicity environment, activity can not be affected, and can not change at uviolizing 24 hours bacteriostatic activities.High temperature test also shows in addition, and high temperature does not still affect effective bacteriostatic ingredients.
Be described in detail specific embodiments of the invention above, but it is just as example, the present invention is not restricted to specific embodiment described above.To those skilled in the art, any equivalent modifications that the present invention is carried out and substituting also all among category of the present invention.Therefore, equalization conversion done without departing from the spirit and scope of the invention and amendment, all should contain within the scope of the invention.

Claims (9)

1. lack a biocontrol microorganisms for sfp gene, it is characterized in that, for bacillus amyloliquefaciens ( bacillus amyloliquefaciens) HAB-2, deposit number is: CCTCC M 2015070.
2. a zymotechnique for biocontrol microorganisms according to claim 1, is characterized in that, is inoculated in by biocontrol microorganisms according to claim 1 in the bacteriolyze broth culture of liquid, and under the condition of 26 ~ 30 DEG C, 50 ~ 500 revs/min are shaken bacterium 1 ~ 3 day, namely obtain fermented liquid.
3. zymotechnique according to claim 2, is characterized in that, also comprises pre-treatment step: first biocontrol microorganisms according to claim 1 to be inoculated on bacteriolyze broth culture at 26 ~ 30 DEG C activation culture 1 ~ 6 day.
4. an extracting method for the fermented product activeconstituents of biocontrol microorganisms as claimed in claim 1, is characterized in that, comprise the following steps:
Step 1, adopt the zymotechnique described in claim 2 ~ 3 any one to obtain fermented liquid, then centrifugal removing thalline, obtains without fermented liquid;
Step 2, without fermented liquid n-butanol extraction, gets extraction liquid, and concentrated removing propyl carbinol, to obtain final product.
5. an extracting method for the fermented product activeconstituents of biocontrol microorganisms according to claim 1, is characterized in that, comprise the following steps:
A, adopt the zymotechnique described in claim 2 ~ 3 any one to obtain fermented liquid, then centrifugal removing thalline, obtains without fermented liquid;
B, without fermented liquid salt acid for adjusting pH value to 1.5 ~ 3, leaves standstill at least 3h at 1 ~ 10 DEG C;
C, centrifugal, abandoning supernatant, gets precipitation, to obtain final product.
6. an extracting method for the fermented product activeconstituents of biocontrol microorganisms according to claim 1, is characterized in that, comprise the following steps:
1) adopt the zymotechnique described in claim 2 ~ 3 any one to obtain fermented liquid, then centrifugal removing thalline, obtains without fermented liquid;
2) slowly add saturated ammonium sulphate, protein precipitation, at 1 ~ 10 DEG C, leave standstill at least 3h;
3) to be centrifugally precipitated, drying to be precipitated use afterwards 1/5 ~ 1/20 original fermented solution volume, pH value be 6 ~ 8 Tris-HCl dissolution precipitation, to obtain final product.
7. biocontrol microorganisms according to claim 1 is preparing the application in lipopeptid class inhibiting-bacteria preparation.
8. the application of biocontrol microorganisms according to claim 1 in preparation control fungal diseases of plants preparation.
9. application according to claim 8, it is characterized in that, described fungal diseases of plants is one or more in early blight of tomato, watermelon blight, banana blight, cotton wilt, rubber anthrax, rubber tree rod spore mould defoliation, tea zonate spot, mango anthracnose and/or rubber brown root rot.
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CN104988096B (en) * 2015-07-21 2018-10-26 中国热带农业科学院环境与植物保护研究所 One plant height effect inhibits biocontrol microorganisms Kg2A and its application of sickle-like bacteria and anthrax-bacilus
CN105018380A (en) * 2015-07-21 2015-11-04 中国热带农业科学院环境与植物保护研究所 Bacillus amyloliquefaciens BR25 and culture method and application thereof
CN104988096A (en) * 2015-07-21 2015-10-21 中国热带农业科学院环境与植物保护研究所 Bio-control bacterium Kg2A capable of efficiently inhibiting fusarium and bacillus anthraci and applications thereof
CN105018380B (en) * 2015-07-21 2018-10-26 中国热带农业科学院环境与植物保护研究所 One bacillus amyloliquefaciens BR25 and its cultural method and application
CN105541848A (en) * 2016-01-18 2016-05-04 海南大学 Bactericidal compound and preparation method thereof
CN105557714A (en) * 2016-01-18 2016-05-11 海南大学 Application of compound H2 in preparation of lipopeptide bacteriostatic preparation
CN105541848B (en) * 2016-01-18 2018-05-08 海南大学 A kind of Fungicidal compounds and preparation method thereof
CN106591144B (en) * 2016-12-01 2019-06-11 中国热带农业科学院橡胶研究所 One plant of multifunctional wood trichoderma strain and its application
CN106591144A (en) * 2016-12-01 2017-04-26 中国热带农业科学院橡胶研究所 Multi-functional trichoderma strain and application thereof
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
CN110184304A (en) * 2019-03-27 2019-08-30 海南大学 A kind of bacillus HN-2 compound killing pathogenetic bacteria and preparation method
CN111357771A (en) * 2020-05-12 2020-07-03 海南大学 Biocontrol bacterium preparation for preventing and treating soil-borne diseases
CN111357771B (en) * 2020-05-12 2021-09-28 海南大学 Biocontrol bacterium preparation for preventing and treating soil-borne diseases
CN113875779A (en) * 2021-10-20 2022-01-04 海南大学 Bactericide composition and preparation method thereof
CN113875779B (en) * 2021-10-20 2022-10-28 海南大学 Bactericide composition and preparation method thereof
CN114164120A (en) * 2021-11-18 2022-03-11 海南大学 Microbial preparation for preventing and treating plant diseases and preparation method and application thereof
CN114287446A (en) * 2021-12-10 2022-04-08 海南大学 Biological agent for inducing plants to prevent and treat fungal diseases and preparation method and application thereof
CN115308342A (en) * 2022-10-11 2022-11-08 海南康植肽生物科技有限公司 Method for detecting surfactant

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