CN101008017A - Bacillus subtilis (Ehrenberg)Cohn fermented product and its production method and uses - Google Patents
Bacillus subtilis (Ehrenberg)Cohn fermented product and its production method and uses Download PDFInfo
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Abstract
The invention relates to a bacillus subtilis fermentate, the preparing method and its application as plant pathogeny antimycotic agent. The applied Bacillus subtilis RP528(CGMCC No.1571) is isolated from manila clam in Qingdao Huanghai sea marine site, which grows well in culture medium with salt content being 1- 4%. It is cultured in culture medium prepared with amylaceum, protease leather, yeast powder, calcium carbonate and old sea water for 48 -72 hours, the metabolite of strain RP528 is secreted out of cell, centrifuging to remove bacteria and getting supernatant, which is the fermentation product. The fermentation product is used as plant pathogeny antimycotic agent, characterized by wide spectrum, high efficiency, and thermal stability.
Description
Technical field:
The present invention relates to a kind of bacillus subtilis (Ehrenberg) Cohn fermented product, and the manufacture method of this tunning and as the application of plant pathogenic fungi antiseptic-germicide.
Background technology:
Chemical pesticide is in control or alleviate and played significant role aspect harm that disease, worm, weeds cause, the protection agriculture production.But the toxicity of chemical pesticide is higher, Stability Analysis of Structures, be difficult to degraded, a large amount of backs of using are residual bigger in crop, and inrichment is strong behind the animal edible, and can be penetrated in soil, river or the underground water, cause environmental pollution, reduce soil quality, destroy Agro-ecology, influence the healthy of people.Even more serious is, because some chemical pesticide of life-time service has caused the resistance of insect and germ, has occurred agricultural chemicals in the agriculture production and has used more and more, the phenomenon that disease and pest is more and more serious.Natural product resource with anti-microbial activity is developed, and studying its toxicity and using is current focus always.The microbial source antiseptic-germicide is free from environmental pollution owing to having, and to the person poultry safety, pathogenic bacteria is difficult for producing characteristics such as resistance, so be the current active research of ten minutes in the world field.
As a kind of disease-preventing microbial, subtilis is subject to people's attention just day by day, and many relevant research reports have been arranged at present.
On June 23rd, 2004, disclose one on the Gazette of Patent for Invention and be called the application for a patent for invention of " a kind of plant endogenesis subtilis and preparation thereof ", number of patent application: 02147811.2, publication number: CN 1506455A.Disclose the new bacterial strain BS-2 of a kind of subtilis in this patent application, deposit number is CGMCC No.0819.This bacterial strain BS-2 separates in the capsicum blade, obtains through the induced mutations screening.Bacterial strain BS-2 can initiatively enter in the plant materials under lower bacterial concentration, and can breed and conduction decided at the higher level but not officially announced the growing of plant materials, and plant is had the growth of promotion and prophylaxis effect simultaneously.When the preparation spraying that contains bacterial strain BS-2 is used, anthrax, Chinese cabbage anthrax and the banana anthracnose of capsicum seedling and fruit had protection effect more than 80%.
On February 16th, 2005, disclose one on the Gazette of Patent for Invention and be called the application for a patent for invention of " subtilis (Bacillus subtilis) strain X i-55 and preparation method thereof ", number of patent application: 200410022543.X, publication number: CN 1580238A.Introduced in this patent application a kind of from the primary soil of Xichang City deep layer the new strain X i-55 of subtilis of separation screening, its preservation registration number is: CGMCC No.1070.This strain X i-55 has inhibition effect more than 80% to rice blast fungus, and the field test in 4 years proves, the effect average out to 60% of its control rice blast.
The new bacterial strain of disclosed subtilis all has antibacterial and prophylaxis effect in above-mentioned two patent applications, but they all are to derive from terrestial enviornment but not marine organisms endogenous subtilis, and the pathogenic bacteria kind of control is also very limited.In addition, disclosed new bacterial strain all is to be prepared into bacteria preparation by the method for fermenting to use when being used for controlling plant diseases in above-mentioned two patent applications, and the purpose bacterial strain in the microbial inoculum must reach certain concentration.When the new bacterial strain of described subtilis reaches certain concentration on plant during a large amount of breeding, though can play preventive and therapeutic effect to relevant pathogenic bacteria, but also might suppress or kill other probiotics, so if improper use can cause adverse influence to the eubiosis.Therefore, may there be problem safe in utilization in the above-mentioned microbial inoculum that contains the new bacterial strain of subtilis.
The research that utilizes terrestrial microorganism to prevent and treat phytopathogen has been carried out considerable time, and it is increasing that screening obtains the difficulty of valuable new bacterial strain or new active substance.The living environment of marine microorganism and terrestrial microorganism have very big-difference, according to the applied ecology principle, practise the screening of taking a sample from non-cause of disease with occupying, the success ratio of obtaining pathogenic bacteria antagonism bacterium or antagonistic substance is also higher, and it is very hopeful therefore to screen the agricultural natural drug with desinsection, sterilization, weeding activity from marine microorganism.
Have miscellaneous microorganism in the ocean, but people are to the development and use of marine microorganism less than 1% still, still have a large amount of marine microorganism resources to remain exploitation.The regional environment of ocean exists diversity and singularity, the ecotope of high salt, high pressure, low temperature, low nutrition and unglazed photograph, make marine microorganism generally have unique pathways metabolism and genetic background, thereby possess the ability that produces the active substance that special construction and function are arranged.A lot of marine microorganisms, though similar to known terrestrial microorganism on taxonomy, but, often can produce the biologically active substance that corresponding terrestrial Institute of Micro-biology can not produce owing to adapted to the particular surroundings of ocean.Exist various commensalismes between marine organisms, and extensively have struggle for existence, in the process of struggle for existence, brought up the marine bioactivity material of a large amount of known Buddhist monks in seeking.Studies show that the symbiotic and epiphyte microorganism of marine animal and plant antimicrobial ratio short of money is higher than oceanic sediment, the initial source of marine microorganism, the overwhelming majority from large-scale animals and plants commensalism in ocean or symbiotic marine microorganism.
Utilize biologically active substances such as marine microorganism development of new human microbiotic, antitumor drug, unsaturated fatty acids, enzyme, enzyme inhibitors, VITAMIN, toxin, existing a large amount of report, but the research that marine microorganism is used for the agroforestry field is just just begun.
On November 19th, 2003, disclose one on the Gazette of Patent for Invention and be called the application for a patent for invention of " bacillus subtilis strain and application ", number of patent application: 03121110.0, publication number: CN1456663A.Disclose the new bacterial strain H728 of a kind of subtilis in this patent application, deposit number is CGMCC No.0912.This bacterial strain H728 separation is worn the river coastal seawater from south, and the main raw material of its fermention medium is potato and sucrose, and the centrifugal gained supernatant liquor of its fermented liquid can be used for the control of graw mold of tomato.This bacterium source is in seawater rather than marine organism, and its object of preventing and treating disease is single.
On December 22nd, 2004, disclose one on the Gazette of Patent for Invention and be called the application for a patent for invention of " method of cultivation and the application thereof of source, ocean subtilis insecticidal activity product ", number of patent application: 200310112809.5, publication number: CN 1556195A.According to this patent application introduction, from seawater, to separate and filter out a strain insect is had highly active bacterial strain, the culture supernatant of this bacterial strain has the activity of killing to Agricultural pests such as bollworms.But what this invention was related is the insecticidal activity of this bacterial strain, the problem that does not relate to fungicidal activity, and this application for a patent for invention is not with the core of inventing, promptly separate the bacillus subtilis strain that obtains and deliver the biomaterial preservation that is used for patented procedure, so that the inadequate problem of disclosure of the Invention may occur.
In sum, yet there are no relevant marine shellfish endogenetic bacteria and agricultural thereof the cultivation of antibacterial substance and the relevant report of utilization.
Summary of the invention:
The technical problem to be solved in the present invention is, the existing subtilis that is used to prepare antiseptic-germicide is mainly derived from terrestial enviornment, and the controlling plant diseases kind of antiseptic-germicide is less, and may have problem safe in utilization; Though but the culture controlling plant diseases of source, existing ocean subtilis only works to a kind of plant pathogenic fungi.
In order to solve the problems of the technologies described above, the present invention aims to provide a kind of marine organisms endogenetic bacteria subtilis (Bacillus subtilis) bacterial strain, utilizes this strain culturing to make the plant pathogenic fungi antiseptic-germicide of broad-spectrum high efficacy.For this reason, the present inventor is an indicator with a kind of important phytopathogen one mango greyness germ (Pestalotiopsis mangiferae) in the agroforestry, adopt conventional dull and stereotyped diffusion process, near the Huanghai Sea marine site intravital marine microorganism of bivalve shellfish picking up from Qingdao has been carried out separation and bioactivity screening, obtained a strain and derived from the intravital subtilis of philippine clam whelp (Bacillussubtilis) RP528 (hereinafter to be referred as bacillus RP528).This bacterial strain RP528 on December 20th, 2005 in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, deposit number is: CGMCC No.1571.
The invention provides a kind of bacillus subtilis (Ehrenberg) Cohn fermented product, this tunning can obtain with following method: fermentation culture subtilis in liquid fermentation medium (Bacillus subtilis) RP528 (CGMCC No.1571), cultivate the meta-bolites of described subtilis RP528 in the substratum, behind centrifugal removal subtilis RP528, the supernatant liquor that obtains containing meta-bolites is tunning.
The above-mentioned bacillus subtilis (Ehrenberg) Cohn fermented product of the present invention, its substratum transfers to pH7.0～8.0, and fermentation is under aerobic conditions carried out, and leavening temperature is 28～30 ℃, and bacterial strain RP528 inoculum size is 1～5% a seed liquor (10 of substratum total amount
7～10
8Cell/mL), fermented incubation time is 48～72h.
Bacterial strain RP528 is in the fermentation culture process, and the excretory meta-bolites is the anti-microbial activity product, and this anti-microbial activity product is secreted in extracellular substratum, when bacterial strain RP528 growth reaches stationary phase, the antibacterial product activity is the strongest in the substratum, and this active result is dissolvable in water in the water, to thermally-stabilised.
Effective constituent in the above-mentioned tunning mainly is the meta-bolites of bacterial strain RP528, adopt the anti-microbial activity difference of the meta-bolites in the tunning that different substratum obtains, through a large amount of experiment sievings, the substratum of tunning of the present invention is mainly formulated with glucose, peptone, yeast powder, lime carbonate and Chen Haishui.
The component concentration of the substratum of the above-mentioned tunning of the present invention is:
Glucose 0.5%～1.5%
Peptone 0.7%～1.5%
Yeast powder 0.3%～1.0%
Lime carbonate 0.1%～0.8%
Chen Haishui 95.2%～98.4%
Above-mentioned all percentage ratios are weight percentage.
The best composition content of the substratum of the above-mentioned tunning of the present invention is:
Glucose 1.0%
Peptone 1.0%
Yeast powder 0.5%
Lime carbonate 0.3%
Chen Haishui 97.2%
Above-mentioned all percentage ratios are weight percentage.
The present invention provides a kind of manufacture method of bacillus subtilis (Ehrenberg) Cohn fermented product again, and this method comprises following sequential steps:
(1) obtaining liq fermention medium;
(2) above-mentioned substratum is transferred to pH7.0～8.0, sterilization back inoculation subtilis (Bacillus subtilis) RP528 (CGMCC No.1571), 150～250r/min shaking table shaking culture, 48～72h, fermentation is under aerobic conditions carried out, leavening temperature is 28～30 ℃, and the meta-bolites of subtilis described in the fermenting process is secreted in extracellular substratum;
(3) from through the described subtilis of centrifugal removal the fermented liquid after step (2) fermentation, the supernatant liquor that obtains containing meta-bolites is tunning.
In the manufacture method of the above-mentioned bacillus subtilis (Ehrenberg) Cohn fermented product of the present invention, the component concentration of substratum is:
Glucose 0.5%～1.5%
Peptone 0.7%～1.5%
Yeast powder 0.3%～1.0%
Lime carbonate 0.1%～0.8%
Chen Haishui 95.2%～98.4%
Above-mentioned all percentage ratios are weight percentage.
In the manufacture method of the above-mentioned bacillus subtilis (Ehrenberg) Cohn fermented product of the present invention, the best composition content of substratum is:
Glucose 1.0%
Peptone 1.0%
Yeast powder 0.5%
Lime carbonate 0.3%
Chen Haishui 97.2%
Above-mentioned all percentage ratios are weight percentage.
In the manufacture method of the above-mentioned bacillus subtilis (Ehrenberg) Cohn fermented product of the present invention, the inoculum size of subtilis in the fermention medium (Bacillus subtilis) RP528 (CGMCC No.1571) is 1～5% a seed liquor (10 of substratum total amount
7～10
8Cell/mL).
The present invention also provides the application of a kind of above-mentioned bacillus subtilis (Ehrenberg) Cohn fermented product as the plant pathogenic fungi antiseptic-germicide.
Tunning of the present invention mainly is the application as camellia anthrax bacteria (Colletotrichum gloeosporioides), mango greyness germ (Pestalotiopsismangiferae), damping off of pine seedlings bacterium (Fusarium oxysporum), apple rot pathogen (Valsaceratosperma), sphaeropsis sapinea bacterium (Sphaeropsis sapinea), kind real go rotten germ (Aspergillus niger) and Chinese rose brown patch germ (Cercospora rosicola) antiseptic-germicide as the application of plant pathogenic fungi antiseptic-germicide.
The bacterial strain RP528 that the present invention adopts separates the philippine clam whelp (Ruditapesphilippinesis) from the coastal waters, Qingdao, and the bacterial strain BS-2 in No. 02147811.2 patent application separates from the capsicum blade, 200410022543.X the strain X i-55 in number patent application separates in the primary soil of Xichang City deep layer, wear the river coastal seawater 03121110.0 the bacterial strain H728 in number patent application separates from south, the bacterial strain RP528 that the present invention adopts is the new bacterial strain of a kind of subtilis (Bacillus subtilis).In the bacillus subtilis (Ehrenberg) Cohn fermented product of the present invention owing to contain the meta-bolites of bacterial strain RP528, can be used as the plant pathogenic fungi antiseptic-germicide uses, show through a large amount of indoor bioassay, it is to mango greyness germ (Pestalotiopsis mangiferae), camellia anthrax bacteria (Colletotrichumgloeosporioides), sphaeropsis sapinea bacterium (Sphaeropsis sapinea), damping off of pine seedlings bacterium (Fusarium oxysporum), apple rot pathogen (Valsa ceratosperma), plant real mildew and rot germ (Aspergillus niger) and Chinese rose brown patch germ (Cercospora rosicola) extraordinary fungistatic effect is all arranged, with respect to microbial antibacterial agent in the above-mentioned patent application, bacillus subtilis (Ehrenberg) Cohn fermented product of the present invention has efficiently as the plant pathogenic fungi antiseptic-germicide, the characteristics of wide spectrum.Bacillus subtilis (Ehrenberg) Cohn fermented product of the present invention can not cause environmental pollution to environment and non-target organism safety.In addition, tunning of the present invention does not have considerable change to hot quite stable even handle its bacteriostatic activity of 30min down at 80 ℃ yet.Owing to adopted bacterial strain RP528 in the manufacture method of bacillus subtilis (Ehrenberg) Cohn fermented product of the present invention, the raw material that bacterial strain RP528 produces the substratum of anti-microbial activity meta-bolites obtains easily, fermentation condition is simple, low for equipment requirements, and prepare substratum with seawater, the osmotic pressure height is difficult for by other living contaminantses in the fermenting process.
The new bacterial strain RP528 of the subtilis that the present invention adopts adopts following method separation screening to obtain.
Isolation medium is formed: peptone 0.5g,, yeast extract paste 0.1g, tertiary iron phosphate 0.01g, agar 1.5g, Chen Haishui 100mL.Isolation medium is transferred to pH7.6～7.8.For separatory marine products bivalve shellfish is philippine clam whelp (Ruditapes philippinesis), mussel (Mytilus edulis), the razor clam of hanging (Sinonovacula constricta) and bay scallop (Argopecten irrdians), all is collected near Huanghai Sea marine site, Qingdao.Whole mask work is finished on Bechtop.During separation, carefully break shell into two with one's hands for the examination shellfish, wash body surface repeatedly with aseptic seawater, carry out surface sterilization with alcohol swab again, with the sterilization scalper digestive tube is cut open, dip in digestive tube with transfering loop and to get a ring, line separates on the solid plate of isolation medium, is inverted into 28 ℃ of incubators and cultivates.Cultivate after 1-5 days, observe and the dissimilar bacterium colony of picking, the separation and purification of further ruling, numbering and going on the isolation medium inclined-plane during for single bacterium colony to be determined is stored under 4 ℃, treats next step screening.Isolate 134 strain marine organisms endogenetic bacterias altogether by above-mentioned steps.
Seed culture medium is formed: glucose 1%, peptone 1%, yeast extract paste 0.5%, lime carbonate 0.3%, Chen Haishui 97.2%.Substratum transfers to pH7.0-8.0, and 20min sterilizes under 121 ℃ of conditions.Choose single colony inoculation in above-mentioned seed culture medium, under 28 ℃ of conditions, 180r/min shaking table shaking culture is spent the night and is seed liquor.
Fermention medium is formed: with the seed substratum.Fermention medium transfers to pH7.0-8.0, and 20min sterilizes under 121 ℃ of conditions.With above-mentioned isolating 134 strain marine organisms endogenetic bacterias, be inoculated in the fermention medium separately, under 28 ℃ of conditions, 180r/min shaking table shaking culture 24～48h.Inoculum size is equivalent to 1% seed liquor (10 of substratum total amount
7～10
8Cell/mL).The centrifugal 10min of fermented liquid 10000r/min removes thalline, gets supernatant liquor for surveying anti-microbial activity.
All percentage ratios in above-mentioned isolation medium, seed culture medium and the fermention medium are weight percentage.
Adopt dull and stereotyped diffusion sieve method, promptly be incubated on the flat board at mango greyness germ (Pestalotiopsis mangiferae) the colony edge 1cm place on the PDA flat board in distance, beat the hole that diameter is 0.4cm with aseptic punch tool, in the hole, add the fermenation raw liquid supernatant 20 μ L after centrifugal, each bacterial strain repeats 3 times, makes blank with the fermention medium that does not connect bacterium that adds equivalent in the hole, puts in 28 ℃ of incubators and cultivates, observations behind the 48h, and measure antibacterial circle diameter.
From 134 strain marine organisms endogenetic bacterias, filter out the very strong marine microorganism of a strain anti-mycotic activity through flat board diffusion sieve method, it is numbered RP528, this bacterial strain RP528 separates from philippine clam whelp (Ruditapes philippinesis), its fermented liquid stoste can reach 13.5mm to mango greyness germ loop diameter, as shown in Figure 1, A1, A2, A3 are treatment group among the figure, and B1, B2, B3 are control group.
Bacterial strain RP528 cell is shaft-like, Dan Sheng, twin or formation short chain, and gram positive, size is 0.8-1.0 * 3-5 μ m, the oval gemma of giving birth in the generation, sporangium is not expanded, and the tool peritrichous can move.The bacterium colony canescence, circle, thickness, projection, surperficial shrinkage.The catalase positive utilizes glucose, pectinose and seminose to produce acid, hydrolyzable casein, gelatin and Zulkovsky starch, VP reacting positive.Can not decompose tyrosine, the lecithinase feminine gender, the phenylalanine deaminase feminine gender can be utilized Citrate trianion, and (G+C) mol% is 44.4.This bacterial strain is a well-grown in 1%～4% the substratum in saltiness.
According to the gramstaining reaction of bacterial strain, cellular form, cultural characteristic, biochemical reactions and (G+C) mol% index etc., with reference to " uncle's outstanding Bacteria Identification handbook (the 8th edition), " common bacteria system identification handbook " and documents such as " study courses of microbiology experimental technique " are accredited as subtilis (Bacillus subtilis) with bacterial strain RP528 of the present invention.
The bacterial strain RP528 that separation screening of the present invention obtains is being positioned at China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation of Beijing on December 20th, 2005, and deposit number is: CGMCC No.1571.
Description of drawings:
Fig. 1 is the antibacterial loop graph that the tunning of bacterial strain RP528 of the present invention occurs mango greyness germ on the PDA flat board;
Fig. 2 is under the different fermentations time conditions, the tunning activity change graphic representation of bacterial strain RP528 of the present invention;
After Fig. 3 was treatment of different temperature, the tunning anti-microbial activity of bacterial strain RP528 of the present invention changed column diagram.
Embodiment:
Embodiments of the present invention is described in detail below in conjunction with accompanying drawing.
Embodiment 1:
Present embodiment is to adopt the new bacterial strain RP528 of subtilis of the present invention to make the method for tunning.This method comprises following sequential steps:
(1) obtaining liq fermention medium, the consisting of of this fermention medium (prescription one): glucose 1.0%, peptone 1.0%, yeast extract paste 0.5%, lime carbonate 0.3%, Chen Haishui 97.2%.
(2) adopt 1mol/L sodium hydroxide to transfer to pH7.0～8.0 above-mentioned fermention medium, 20min sterilizes under 121 ℃ of conditions.Subtilis (Bacillus subtilis) RP528 (CGMCCNo.1571) is inoculated in the fermention medium after the sterilization, and inoculum size is equivalent to 1% seed liquor (10 of substratum total amount
7～10
8Cell/mL), under 28 ℃ of conditions, 180r/min shaking table shaking culture 48h.Fermentation is under aerobic conditions carried out, and the meta-bolites of the RP528 of subtilis described in the fermenting process is secreted in extracellular substratum.
(3) will remove subtilis RP528 by centrifugal 10min through the fermented liquid 10000r/min after step (2) fermentation, the supernatant liquor that obtains containing meta-bolites is tunning.
The tunning that obtains is faint yellow, and pH is 5.5～6.0.
Embodiment 2:
The step of present embodiment is identical with embodiment 1, just changes the fermented incubation time in the step (2) among the embodiment 1 into 24h.
Embodiment 3:
The step of present embodiment is identical with embodiment 1, just changes the fermented incubation time in the step (2) among the embodiment 1 into 72h.
Embodiment 4:
The step of present embodiment is identical with embodiment 1, just changes the fermented incubation time in the step (2) among the embodiment 1 into 96h.
Embodiment 5:
The step of present embodiment is identical with embodiment 1, and just the composition with the fermention medium in the step (1) among the embodiment 1 changes (prescription two) into: Zulkovsky starch 2%, analysis for soybean powder 1.5%, peptone 0.2%, yeast extract paste 0.5%, lime carbonate 0.3%, Chen Haishui 95.5%.
Embodiment 6:
The step of present embodiment is identical with embodiment 1, and just the composition with the fermention medium in the step (1) among the embodiment 1 changes (prescription three) into: potato juice 2%, sucrose 2%, lime carbonate 0.3%, Chen Haishui 95.7%.
(Pestalotiopsis mangiferae) is indicator with mango greyness germ, the tunning that obtains among the foregoing description 1, embodiment 2, embodiment 3, embodiment 4, embodiment 5 and the embodiment 6 carried out anti-microbial activity measure.Concrete measuring method adopts dull and stereotyped diffusion process, promptly be incubated on the flat board at mango greyness germ (Pestalotiopsis mangiferae) the colony edge 1cm place on the PDA flat board in distance, beat six holes that diameter is 0.4cm with aseptic punch tool, in six holes, add the tunning 20 μ L that obtain among the foregoing description 1, embodiment 2, embodiment 3, embodiment 4, embodiment 5 and the embodiment 6 respectively, put in 28 ℃ of incubators and cultivate, observations behind the 48h, and measure antibacterial circle diameter.Said determination is triplicate altogether, calculates average diameter of inhibition zone, the relation that obtains fermentation time and tunning anti-microbial activity as shown in Figure 2, the relation that obtains different substratum and tunning anti-microbial activity is as shown in table 1.
Table 1:
| Substratum | Average diameter of inhibition zone (mm) |
| Fill a prescription two the prescription three | 13.5 10.4 6.3 |
After the tunning anti-microbial activity that above-mentioned 6 embodiment are obtained compares, can determine that the optimal conditions of fermentation of bacterial strain RP528 is:
1. fermentative medium formula: glucose 1%, peptone 1%, yeast extract paste 0.5%, lime carbonate 0.3%, Chen Haishui 97.2%, all percentage ratios are weight percentage.
2. fermention medium pH transfers to 7.0～8.0, and inoculum size is 1% a seed liquor (10 of substratum total amount
7～10
8Cell/mL).
3. under 28 ℃ of conditions, 180r/min shaking table shaking culture 48h.
Embodiment 7:
Present embodiment is the application of tunning of the present invention as the plant pathogenic fungi antiseptic-germicide.The tunning that obtains among the embodiment 1 as antibiotic aqua, is used for the Plant diseases that plant pathogenic fungi causes is prevented and treated.Adopt above-mentioned dull and stereotyped diffusion process that the tunning that obtains among the embodiment 1 has been carried out antimicrobial spectrum mensuration, the test plant pathogenic fungi is: camellia anthrax bacteria (Colletotrichumgloeosporioides), mango greyness germ (Pestalotiopsis mangiferae), damping off of pine seedlings bacterium (Fusarium oxysporum), apple rot pathogen (Valsa ceratosperma), sphaeropsis sapinea bacterium (Sphaeropsis sapinea), real germ (Aspergillus niger) and the Chinese rose brown patch germ (Cercospora rosicola) of going rotten of kind.Each calculates average antibacterial circle diameter for examination fungi repeated test 5 times, obtains tunning among the embodiment 1 as the antimicrobial spectrum result such as the table 2 of sterilization aqua,
Table 2
| Test plant pathogenic fungi kind | Antibacterial circle diameter (mm) |
| Camellia anthrax bacteria (Colletotrichum gloeosporioides) mango greyness germ (Pestalotiopsis mangiferae) | 13.2 13.4 |
| Damping off of pine seedlings bacterium (Fusarium oxysporum) apple rot pathogen (Valsa ceratosperma) Sphaeropsis sapinea (Sphaeropsis sapinea) is planted real germ (Aspergillus niger) the Chinese rose brown patch germ (Cercospora rosicola) that goes rotten | 11.2 11.6 8.8 9.4 10.2 |
From the result of table 2 as can be seen, tunning of the present invention has characteristics efficient, wide spectrum as the plant pathogenic fungi antiseptic-germicide.
Embodiment 8:
Present embodiment is that tunning of the present invention is as the application of plant pathogenic fungi antiseptic-germicide in control camellia anthrax.Healthy anosis potted plant camellia seedling 16 strains of choosing, every basin one strain about plant height 0.5m, is divided into two groups, every group 8 basin, one group of treatment group for the sprinkling tunning, another group is control group.Whenever 10 blades of selecting good strains in the field for seed, selected blade size, quality and adnation position should be close as far as possible between each strain.Earlier the tunning that obtains among the embodiment 1 is sprayed at equably with atomizer on the blade of treatment group, spray inoculation anthrax pathogenic bacteria conidial suspension behind the natural air drying, spore concentration is 1 * 10
6Individual/mL, inoculation back humidification cotton balls entangles the plant 48h that preserves moisture with plastics bag, and the control group blade sprays the fermention medium (prescription one) that does not connect bacterium, spray inoculation anthrax pathogenic bacteria conidial suspension behind the natural air drying, and spore concentration is 1 * 10
6Individual/mL, use with the quadrat method 48h that preserves moisture.Behind the 48h treatment group is sprayed the tunning that obtains among embodiment 1 again, put in 25～28 ℃ of greenhouses, the routine observation and the record state of an illness, statistical computation protection effect after 20 days.Lesion area according to each blade is divided into Pyatyi with the state of an illness.1 grade is no scab; 2 grades is lesion area＜5%; 3 grades is lesion area＜5-20%; 4 grades is lesion area＜20-50%; 5 grades is lesion area＞50%.Every processing blade amts at different levels are A, B, C, D, E, and then every processing sickness rate accounts for the percentage ratio of total investigation number of sheets for this processing incidence of leaf number; Disease index=(0 * A+1 * B+2 * C+3 * D+4 * E)/(4 * (A+B+C+D+E)) * 100; Protection effect=100%-(the average disease index of the average disease index/control group of treatment group) * 100%.Experimental result sees Table 3, and the tunning that obtains among the embodiment 1 can reach 85.9% to the prevention effect of camellia anthrax.
Table 3:
| Project | Leaf sickness rate (%) | Disease index | Prevention effect (%) |
| The treatment group control group | 18.8 95.0 | 12.3 87.2 | 85.9 |
Embodiment 9:
Present embodiment is the thermostability experiment of tunning of the present invention.The tunning 1mL that obtains among the embodiment 1 is packed in four Eppendorf pipes, put respectively at 30 ℃, 50 ℃, 70 ℃, 80 ℃ following constant temperature and handle 30min, adopt the bacteriostatic activity of above-mentioned dull and stereotyped diffusion process detection to mango greyness germ then, detected result is seen Fig. 3.Experimental result shows that the tunning that obtains among the embodiment 1 does not have considerable change to hot quite stable even handle its bacteriostatic activity of 30min down at 80 ℃ yet
Claims (10)
1. bacillus subtilis (Ehrenberg) Cohn fermented product, this tunning can obtain with following method: (Bacillus subtilis) RP528 of fermentation culture subtilis in liquid fermentation medium (CGMCCNo.1571), cultivate the meta-bolites of described subtilis RP528 in the substratum, behind centrifugal removal subtilis RP528, the supernatant liquor that obtains containing meta-bolites is tunning.
2. tunning according to claim 1 is characterized in that: described substratum is mainly formulated with glucose, peptone, yeast powder, lime carbonate and Chen Haishui.
3. tunning according to claim 2 is characterized in that: the component concentration of substratum is:
Glucose 0.5%～1.5%
Peptone 0.7%～1.5%
Yeast powder 0.3%～1.0%
Lime carbonate 0.1%～0.8%
Chen Haishui 95.2%～98.4%
Above-mentioned all percentage ratios are weight percentage.
4. tunning according to claim 3 is characterized in that: the best composition content of substratum is:
Glucose 1.0%
Peptone 1.0%
Yeast powder 0.5%
Lime carbonate 0.3%
Chen Haishui 97.2%
Above-mentioned all percentage ratios are weight percentage.
5. the manufacture method of a bacillus subtilis (Ehrenberg) Cohn fermented product, this method comprises following sequential steps:
(1) obtaining liq fermention medium;
(2) above-mentioned substratum is transferred to pH7.0～8.0, sterilization back inoculation subtilis (Bacillus subtilis) RP528 (CGMCC No.1571), 150～250r/min shaking table shaking culture, 48～72h, fermentation is under aerobic conditions carried out, leavening temperature is 28～30 ℃, and the meta-bolites of subtilis described in the fermenting process is secreted in extracellular substratum;
(3) from through the described subtilis of centrifugal removal the fermented liquid after step (2) fermentation, the supernatant liquor that obtains containing meta-bolites is tunning.
6. method according to claim 5 is characterized in that: the component concentration of substratum is:
Glucose 0.5%～1.5%
Peptone 0.7%～1.5%
Yeast powder 0.3%～1.0%
Lime carbonate 0.1%～0.8%
Chen Haishui 95.2%～98.4%
Above-mentioned all percentage ratios are weight percentage.
7. method according to claim 6 is characterized in that: the best composition content of substratum is:
Glucose 1.0%
Peptone 1.0%
Yeast powder 0.5%
Lime carbonate 0.3%
Chen Haishui 97.2%
Above-mentioned all percentage ratios are weight percentage.
8. according to claim 5,6 or 7 described methods, it is characterized in that: the inoculum size of subtilis in the fermention medium (Bacillus subtilis) RP528 (CGMCC No.1571) is 1～5% a seed liquor (10 of substratum total amount
7～10
8Cell/mL).
9. claim 1,2,3 or 4 described tunnings are as the application of plant pathogenic fungi antiseptic-germicide.
10. tunning according to claim 9 is characterized in that as the application of plant pathogenic fungi antiseptic-germicide: mainly be as camellia anthrax bacteria (Colletotrichum gloeosporioides), mango greyness germ (Pestalotiopsis mangiferae), damping off of pine seedlings bacterium (Fusariumoxysporum), apple rot pathogen (Valsa ceratosperma), sphaeropsis sapinea bacterium (Sphaeropsis sapinea), plant the application of real go rotten germ (Aspergillus niger) and Chinese rose brown patch germ (Cercospora rosicola) antiseptic-germicide.
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| CN104109643A (en) * | 2014-04-23 | 2014-10-22 | 中国海洋大学 | Bacillus spp with broad-spectrum antibacterial activity |
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| CN104109643A (en) * | 2014-04-23 | 2014-10-22 | 中国海洋大学 | Bacillus spp with broad-spectrum antibacterial activity |
| CN104195061A (en) * | 2014-04-23 | 2014-12-10 | 河南科技大学 | Bacillus subtilis and application thereof |
| CN104195061B (en) * | 2014-04-23 | 2017-01-18 | 河南科技大学 | Bacillus subtilis and application thereof |
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| CN111670914A (en) * | 2020-07-06 | 2020-09-18 | 南京林业大学 | Novel application of bacillus pumilus HR10 in prevention and treatment of forest diseases |
| CN111670914B (en) * | 2020-07-06 | 2021-04-13 | 南京林业大学 | Novel application of bacillus pumilus HR10 in prevention and treatment of forest diseases |
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