CN104212723A - Artemisia japonica endophytic pestalotiopsis uvicola and application thereof - Google Patents
Artemisia japonica endophytic pestalotiopsis uvicola and application thereof Download PDFInfo
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Plant Substances (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to the technical field of microorganisms. An artemisia japonica endophytic fungus is collected from living bodies of artemisia japonica from a suburb of Guiyang city in Guiyang province through endophytic fungus separation technology. The artemisia japonica endophytic fungus is named pestalotiopsis uvicola GMH 31 through authentication by microbial taxonomy. The strain is deposited in China center for type culture collection with a deposition date being June-30-2014 and an accession number being CCTCC No: M2014303. The invention has following most significant characteristics: (1) A fermentation liquid of the strain is an active metabolite which has inhibiting effects of sclerotinia sclerotiorum, phytophthora capsici and the like and is an important microbial resource; and (2) A fermentation mycelium of the strain can secret various natural active components, has medicinal effects of brad-spectrum tumor resistance and the like, and is an important microbial resource of a novel natural active drug.
Description
Technical field
The present invention relates to microbial technology field, especially relate to the outstanding Wei of plan Pestalotia endogenetic fungus that a strain derives from medicinal plant Artemisia japonica and can intend coiling stey (Pestalotiopsis uvicola) GMH31 and the application of meta-bolites in agricultural and medicine thereof.
Background technology
Plant endogenesis epiphyte (fungal endophyte) refers to the certain phase of the life history or all stage life and health plant respectively organize qualified intraorganic fungi.They and host plant are by defining symbiotic relationship closely mutually, and can promote the growth of plant, provide plant to tackle the adaptive faculty of coercing, some plant endogenesis epiphytes also have pharmaceutical use.Find from plant endogenesis epiphyte and find that active compound has become the another focus of research both at home and abroad.As Chinese patent literature CN201310194836.5 discloses in a strain rough gentian endogenetic fungus (Metarrhizium) LD421 being separated and obtaining, can be used for control rough gentian leaf blight; Patent application CN201210067829.4 then discloses and be separated the endogenetic fungus fusarium solani T-7 obtained from the tissue such as Radix Ginkgo, stem, leaf, has good restraining effect to P. capsici, tomato wilt bacterium, Valsa mali etc.
Artemisia japonica (Artemisia japonica) is composite family artemisia, its bitter, micro-sweet, cool in nature.Among the people careless also known as soil purple (" Luchuan draft ") recklessly, stomachache spirit, bear's paw, with all herbal medicine.There is heat-clearing, cool blood, removing toxic substances, stomach invigorating hemostasia effect, be mainly used in treating cold, fever, heatstroke, malaria, pulmonary tuberculosis hectic fever, essential hypertension, infirmities and diseases, sphincter disturbance and venomous snake bite etc.Simultaneously also Artemisia japonica on the books is medicine food dual purpose plant, containing various active material, has immunity, antiviral, antitumor, hypoglycemic, reducing blood-fat protects the liver and to delay senility etc. different physiological roles.In addition, Artemisia japonica also can be used for preparing biological pesticide, uses as sterilant.
The Main Means controlling crop diseases and pest crop smothering at present in agricultural production process is chemical pesticide control, it is to alleviating disease pest and weed, ensure that the good harvest of farm crop high yield plays a positive role, but due to the mutual restriction interdependence between nature biotechnology, do not advise now too relying on chemical pesticide in the process carrying out disease.Therefore, to the research of plant endogenesis epiphyte, utilizing the relation of plant endogenesis epiphyte and the symbiosis of host plant biological activity, screen the endogenetic fungus with high biological activity, obtain active result further, is the large focus studied this year.But, for a certain host plant, which kind of endogenetic fungus or which class endogenetic fungus have resisting pathogenic microbes activity actually, and which kind of bacterium activity is the highest in each endogenetic fungus, whether separation and purification is easy, whether fermenting process is complicated etc., all needs to carry out large quantity research and confirmed, not yet finds that Artemisia japonica endogenetic fungus and separation and Culture and resisting pathogenic microbes thereof are active, the concrete report of medical active application at present.
Summary of the invention
The object of the invention is, for the dangerous deficiency less with microbial source drug variety of pharmaceutical chemicals, to provide a kind of endogenetic fungus separated from medicinal plant Artemisia japonica---You Wei can intend dish stey (Pestalotiopsis uvicola).
Endogenetic fungus of the present invention, be separated to obtain from the medicinal plant Artemisia japonica (Artemisia japonica) in suburb, Guiyang City, Guizhou Province, called after Nei Shengyouwei can intend coiling stey (Pestalotiopsis uvicola) GMH31, and has carried out multiple bioactivity research to the fermentating metabolism product of this bacterial strain.This invention for microbial pesticide and medicine further Study and Development excellent starting strain is provided.
Technical solution of the present invention is as follows:
The outstanding Wei of the present invention can intend the separation, the selection systems that coil stey:
Be in the Artemisia japonica blade gathered in suburb, Guiyang City, Guizhou Province in August, 2011, obtain through steps such as separation, purifying, cultivation, fermentation and determinations of activity and preserve; Be accredited as You Wei through microbial taxonomy and can intend dish stey (Pestalotiopsis uvicola), name as You Wei can intend coiling stey (Pestalotiopsis uvicola) GMH31.This bacterial strain in China typical culture collection center preservation, preservation date on June 30th, 2014, preservation registration number CCTCCNO:M2014303, Luojiashan, Wuchang, Wuhan City, Hubei Province, address.
Concrete isolation cultivation method is:
(1) preparation of Japanese wormwood herb botanical tissue: the leaf texture gathering Artemisia japonica, with tap water, the raw microorganism of table of removing sample surfaces, is then put in wind on clean gauze and dries;
(2) sterilization and Sterility testing is shown: the tissue block after drying is cut into suitable size, rinses well with sterile purified water, blade sterilization employing 75% alcohol immersion 1-2min, 0.1%HgCl
2solution soaking 3-4min, then rinse to thimerosal noresidue with sterile purified water, after blotting tissue segments remained on surface water droplet with sterilizing filter paper, what be cut into diameter 4 ~ 6mm size organizes segment;
(3) separation and purification of endogenetic fungus: the segment of organizing after step (2) being sterilized is inoculated in containing Vetstrep (100 μ gmL
-1) and penicillin (50 μ gmL
-1) PDA culture medium flat plate on, in 25-30 DEG C of constant temperature culture 2 ~ 7 days after sealing, grow from tissue block to mycelium, adopt Tip Splitting picking method, mycelia is transferred on purifying PDA culture medium flat plate, at 25-30 DEG C, carry out purifying cultivate 5 ~ 9 days, grow complete bacterium colony, be Artemisia japonica endogenetic fungus.
Preferably, in described step (3), culture temperature is 28 DEG C, and slat chain conveyor, purifying incubation time is 7 days.
The outstanding Wei of this Artemisia japonica endogenetic fungus called after can intend dish stey GMH31, its solid culture is characterized as: bacterium colony on PDA flat board 28 DEG C cultivate that within 8-9 days, to extend to diameter be 9cm, bacteria colony white flocculence, colyliform expansion is not obvious, the light safran in the back side, without wheel line, sporophore prepared Chinese ink shape, particle is comparatively large, distributes more sparse.Micro-morphology: acervulus black, closely spherical, under just burying raw Cuticle, break through epidermis after ripe and expose, in grain point-like; Conidium 5 cell, two terminal cells colourless (conically or trilateral), intermediate cell is olive colour to brown (9.7 ~ 12.6 μm), nearly fusiform, uprightly, and wall thickness, size is 19.7 ~ 23.5 × 5.5 ~ 6.6 μm; Appendage and is born in conidial top, is 2 ~ 3, and angle is opened a business, and more elongated, length is 5.9 ~ 6.5 μm, and bottom is colourless, trilateral, the short or nothing of tailfiber.See Fig. 1, Fig. 2.
The molecular biological characteristics of bacterial strain GMH31: adopt molecular biology round pcr, determined dna sequence analysis, the ITSrDNA genome of bacterial strain GMH31 is made up of 495 bases (bp).Phylogenetic tree construction based on ITSrDNA sequence, this bacterium is that You Wei can intend coiling stey, sees Fig. 3.
Another object of the present invention is to provide the method utilizing this bacterial strain fermentation liquor meta-bolites to prevent and treat fungal diseases of plants.
Above-mentioned purpose is achieved through the following technical solutions:
Prevent and treat a preparation method for the microbial metabolites of the fungal diseases of plants such as Kiwifruit brown heart and capsicum epidemic disease, carry out according to the following steps:
(1) activation culture of bacterial classification: the outstanding Wei being separated preservation can be intended dish stey GMH31 bacterial classification and move on potato glucose PDA solid plate, cultivate under 28 ± 1 DEG C of conditions, covering whole culture dish to mycelia, is that the punching of 7mm punch tool obtains bacterium cake, for inoculation with diameter;
(2) liquid fermentation and culture: fermentation culture is containing Semen Maydis powder 10g, N.F,USP MANNITOL 20g, glucose 20g, yeast extract paste 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 500mL triangle; Prepare rear 121 DEG C of sterilizing 20-30min, to be cooled to 40 DEG C aseptically picking 1 diameter be 7mm bacterium cake, under 28 ± 1 DEG C of conditions, standing for fermentation cultivate 28-35 days;
(3) broth extraction: collect fermented liquid after fermentation, adds the extraction into ethyl acetate of 1-3 times of volume, takes off a layer solution, again with the n-butanol extraction of 1-2 times of volume, get upper liquid after layering, be concentrated into medicinal extract under vacuum, obtain propyl carbinol fermented liquid extract.
Another object of the present invention is to provide this strain fermentation mycelium meta-bolites in the antitumor application waited in medicine of preparation.
Above-mentioned purpose is achieved through the following technical solutions:
There is a preparation method for the mycelium meta-bolites of anti-tumor activity, carry out according to the following steps:
(1) activation culture of bacterial classification: the outstanding Wei being separated preservation can be intended dish stey GMH31 bacterial classification and move on potato glucose PDA solid plate, cultivate under 28 ± 1 DEG C of conditions, covering whole culture dish to mycelia, is that the punching of 7mm punch tool obtains bacterium cake, for inoculation with diameter;
(2) liquid fermentation and culture: fermentation culture is containing Semen Maydis powder 10g, N.F,USP MANNITOL 20g, glucose 20g, yeast extract paste 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 500mL triangle; Prepare rear 121 DEG C of sterilizing 20-30min, to be cooled to 40 DEG C aseptically picking 1 diameter be 7mm bacterium cake, under 28 ± 1 DEG C of conditions, standing for fermentation cultivate 28-35 days;
(3) mycelium extracts: collect mycelium after fermentation, adding concentration is 30-90% aqueous acetone solution, the concentration of mycelium in aqueous acetone solution is made to be less than or equal to 0.50g/L, ultrasonic extraction 2-5 time under power is 40KHz, the condition of 30min/ time, then under equal conditions add equal-volume ethyl acetate ultrasonic extraction 2-5 time again, filter, collect upper strata ethyl acetate solution, be concentrated into medicinal extract under vacuum, obtain ethyl acetate mycelium extract.
Beneficial effect of the present invention:
One is that the present invention separates metabolite and has Activities of Some Plants fungal disease pathogenic bacteria and tumour cell and can intend dish stey GMH31 compared with the outstanding Wei of high inhibition effect from medicinal plant Artemisia japonica blade, and this be that the developing of agriculturally control fungal diseases of plants pathogenic bacteria and pharmaceutically anti-malignant tumor source new drugs provides a kind of microorganism strains.
Two is the invention provides to utilize outstanding Wei can intend coiling the method that stey GMH31 fermentation obtains active-fermented broth meta-bolites; This meta-bolites can be applicable to the control of fungal diseases of plants, and the exploitation for disinfectant use in agriculture adds new approach.
Three is the invention provides to utilize outstanding Wei can intend coiling the method that stey GMH31 fermentation obtains active bacteria filament meta-bolites; This meta-bolites can be applicable to the preparation of antitumor drug, and the exploitation of newly originating for medical adds new approach.
Accompanying drawing explanation
Fig. 1 is the lithograph that You Wei can intend coiling stey (Pestalotiopsis uvicola) GMH31 bacterial strain.
Fig. 2 is the conidium figure that You Wei can intend coiling stey (Pestalotiopsis uvicola) GMH31 bacterial strain.
Fig. 3 is the gene tree graph that You Wei can intend coiling stey (Pestalotiopsis uvicola) GMH31 bacterial strain.
Embodiment
In order to make those of ordinary skill in the art better understand the present invention, the applicant has carried out series of experiment research, to prove effect of the present invention.
Below, enumerate embodiment and the present invention is further described, but the present invention is not limited to following embodiment.
The outstanding Wei of embodiment 1:(can intend the isolation and screening coiling stey GMH31)
The present invention gathers medicinal plant---the healthy leaves of Artemisia japonica in August, 2011 in suburb, Guiyang City, Guizhou Province, be separated through surface sterilization, endogenetic fungus, cultivate, fermentation, determination of activity and to the step such as screening suppressing fungal diseases of plants pathogenic bacteria active bacterial strain and tumor cell line, therefrom obtain outstanding Wei and can intend dish stey GMH31, preserve.
Concrete isolation cultivation method is: the preparation of (1) Japanese wormwood herb botanical tissue: the leaf texture gathering Artemisia japonica, and with tap water, the raw microorganism of table of removing sample surfaces, is then put in wind on clean gauze and dries;
(2) sterilization and Sterility testing is shown: the tissue block after drying is cut into suitable size, rinses well with sterile purified water, blade sterilization employing 75% alcohol immersion 1min, 0.1%HgCl
2solution soaking 3min, then rinse to thimerosal noresidue with sterile purified water, after blotting tissue segments remained on surface water droplet with sterilizing filter paper, what be cut into diameter 4-6mm size organizes segment;
(3) separation and purification of endogenetic fungus: the segment of organizing after step (2) being sterilized is inoculated in containing Vetstrep (100 μ gmL
-1) and penicillin (50 μ gmL
-1) PDA culture medium flat plate on, in 28 DEG C of constant temperature culture 5 days after sealing, grow from tissue block to mycelium, adopt Tip Splitting picking method, mycelia is transferred on purifying PDA culture medium flat plate, at 28 DEG C, carries out purifying cultivate 7 days, grow complete bacterium colony, be Artemisia japonica endogenetic fungus.
Outstanding Wei can intend dish stey GMH31 Antibacterial Activity:
Learn from else's experience the fermented liquid 1mL of 0.22 μm of membrane filtration in sterile petri dish, the PDA substratum being cooled to 50 DEG C with 9mL mixes rapidly, Kiwifruit brown rot germ (Sclerotinia sclerotiorum) that 1 diameter is 7mm or phytophthora blight of pepper (Phytophthora capsici) etc. are put respectively for examination bacterium bacterium cake in each substratum plane after cooling, bacterium cake is connected to culture dish central authorities (culture dish diameter is 9cm), put 28 DEG C of cultivation 72h in incubator, with sterilized water process in contrast, repeat 3 times; Right-angled intersection method is adopted to measure colony diameter, with following formulae discovery inhibiting rate: I (%)=[(contrast colony diameter-process colony diameter)/(contrast colony diameter-7)] × 100%.
Measurement result shows: You Wei can intend the inhibiting rate of dish stey GMH31 meta-bolites to Kiwifruit brown rot germ and phytophthora blight of pepper and be respectively 82.8% and 86.9%.
Outstanding Wei can intend dish stey GMH31 inhibition tumor cell strain growth measurement:
Tumor cell suspension is inoculated in 96 well culture plates, every hole 100 μ L, after standard conditions cultivate 12h, adds the nutrient solution 50 μ L (concentration is respectively 20 μ g/mL and 200 μ g/mL) containing contrast or fermentation mycelium extract respectively.Standard conditions cultivate 12 respectively, 24,48,72,96h, terminate front 4h in cultivation, each hole adds the MTT solution 50 μ L that concentration is 2mg/mL.After cultivation terminates, abandon supernatant, every hole adds dimethyl sulfoxide (DMSO) (DMSO) 200 μ L termination reaction, in automatic microplate reader, measure the optical density value (OD value) of each hole at wavelength 570nm place, calculate cell inhibitory rate: I (%)=[1-medicine feeding hole OD value/control wells OD value] × 100%.The bacterial strain for having tumor cytotoxic activity of inhibiting rate >50%.
Measurement result shows: concentration is that the outstanding Wei of 20 μ g/mL can intend dish stey GMH31 meta-bolites, when 72h, to Murine Ascitic Hepatoma Cells H
22, mouse hydroperitoneum type cervical cancer cell U
14with mouse hydroperitoneum type sarcoma cell S
180inhibiting rate be respectively 86.1%, 92.4% and 77.9%.
The outstanding Wei of embodiment 2:(can intend the preparation method of coiling stey GMH31 fermentating metabolism product)
Carry out according to the following steps:
(1) activation culture of bacterial classification: the outstanding Wei being separated preservation can be intended dish stey GMH31 bacterial classification and move on potato dextrose agar PDA solid plate, cultivate under 28 ± 1 DEG C of conditions, covering whole culture dish to mycelia, is that the punching of 7mm punch tool obtains bacterium cake, for inoculation with diameter;
(2) liquid fermentation and culture: fermentation culture is containing Semen Maydis powder 10g, N.F,USP MANNITOL 20g, glucose 20g, yeast extract paste 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 300mL triangle; Prepare rear 121 DEG C of sterilizing 20min, to be cooled to 40 DEG C aseptically picking 1 diameter be the bacterium cake of 7mm, under 28 ± 1 DEG C of conditions, standing for fermentation cultivates 30 days;
(3) broth extraction: collect fermented liquid after fermentation, adds equal-volume extraction into ethyl acetate, takes off a layer solution, then uses equal-volume n-butanol extraction, gets upper liquid, be concentrated into medicinal extract under vacuum after layering, obtains propyl carbinol fermented liquid extract;
(4) mycelium extracts: collect mycelium after fermentation, adding concentration is 80% aqueous acetone solution, the concentration of mycelium in aqueous acetone solution is made to be less than or equal to 0.50g/L, ultrasonic extraction 4 times under power is 40KHz, the condition of 30min/ time, then under equal conditions add equal-volume ethyl acetate ultrasonic extraction 4 times again, filter, collect upper strata ethyl acetate solution, be concentrated into medicinal extract under vacuum, obtain ethyl acetate mycelium extract.
It is propyl carbinol fermented liquid extract prepared in embodiment 2 times methods (3) that outstanding Wei described in following examples 3 can be intended coiling stey GMH31 meta-bolites; The percentage composition of described product is mass/volume per-cent.
The outstanding Wei of embodiment 3:(can intend coiling the restraining effect of stey GMH31 fermented liquid meta-bolites to Kiwifruit brown rot germ and phytophthora blight of pepper)
(1) accurately take You Wei and can intend dish stey GMH31 propyl carbinol fermented liquid extract 0.1g, add the sterilized water of 10mL volume, ultrasonic oscillation fully dissolves, and is finally configured to the mother liquor that concentration is 10g/L;
(2) Antibacterial Activity: get above-mentioned mother liquor and be diluted to the solution that concentration is 1000mg/L and 500mg/L respectively, get each strength solution 1mL in sterile petri dish, the PDA substratum being cooled to 50 DEG C with 9mL mixes rapidly (it is 100mg/L and 50mg/L that You Wei can intend coiling stey GMH31 meta-bolites final concentration), put in each substratum plane the Kiwifruit brown rot germ and phytophthora blight of pepper bacterium cake that 1 diameter is 7mm respectively after cooling, bacterium cake is connected to culture dish central authorities (culture dish diameter is 9cm), put 28 DEG C of cultivation 36h in incubator, with conventional chemical medicament and sterilized water process in contrast, repeat 3 times, right-angled intersection method is adopted to measure colony diameter, with following formulae discovery inhibiting rate: I (%)=[(contrast colony diameter-process colony diameter)/(contrast colony diameter-7)] × 100%.
(3) product of the present invention and conventional pesticide prevention effect simultaneous test: test-results is in table 1.As seen from Table 1, it is slightly excellent compared with conventional chemical medicament that You Wei can intend dish stey GMH31 meta-bolites inhibition, and safety.
The outstanding Wei of table 1 can be intended coiling stey GMH31 meta-bolites to the inhibition for examination pathogenic fungi
"-" represents not test (N.T.).
It is ethyl acetate mycelium extract prepared in embodiment 2 times methods (4) that outstanding Wei described in following examples 4 can be intended coiling stey GMH31 meta-bolites; The percentage composition of described product is mass/volume per-cent.
The outstanding Wei of embodiment 4:(can intend dish stey GMH31 fermentation mycelium meta-bolites to Murine Ascitic Hepatoma Cells H
22, mouse hydroperitoneum type cervical cancer cell U
14with mouse hydroperitoneum type sarcoma cell S
180restraining effect)
(1) accurately take You Wei and can intend dish stey GMH31 ethyl acetate mycelium extract, after first using a small amount of dissolve with ethanol, then be mixed with water for injection dilution the solution that concentration is 0.2mg/mL, fill with for mouse and feed administration;
(2) preparation of tumor cell line animal model: respectively by Murine Ascitic Hepatoma Cells H
22, mouse hydroperitoneum type cervical cancer cell U
14with mouse hydroperitoneum type sarcoma cell S
180make 1 × 10
6cell suspension, gets this cell suspension and only injects mouse (t is sheerly 615 mouse, Balb/c mouse, kunming mouse some, body weight 20-22g) abdominal cavity with 0.5ml/.Next day is divided into control group, test group at random.Control group every mouse stomach 1mL physiological saline, the above-mentioned concentration of test group every mouse stomach is the ethyl acetate mycelium extract aqueous solution 1mL of 0.2mg/mL.Continuous gavage 5 days, observes mouse storaging current, calculates survival rate and increase in life span.
(3) the anti-tumor in vivo effect test of product of the present invention: test-results is in table 2.As seen from Table 2, it is better that You Wei can intend dish stey GMH31 meta-bolites Tumor suppression effect, and safety.
The outstanding Wei of table 2 can intend dish stey GMH31 meta-bolites anti-tumor in vivo test-results
Survival rate: observe more than 60 days non-bearing tumors.
The outstanding Wei of embodiment 5:(can intend the animal toxicity test coiling stey GMH31 fermentation mycelium meta-bolites)
(1) acute toxicity test: get kunming mice 40 (male and female half and half), body weight 22-22g, gavage 3 times in 24 hours, the dose added up is made to be above-mentioned medicinal 225 times, Continuous Observation mouse 14 days, without the phenomena of mortality and untoward reaction, illustrate that this fermentation mycelium meta-bolites has no side effect.
(2) long term toxicity test: get the rat 40 (male and female half and half) that body weight is suitable, be divided into two groups at random, one group was that You Wei can intend dish stey GMH31 fermentation mycelium meta-bolites test group, with 100 times of above-mentioned routine test dosage continuous gavages 60 days; Another group, for adding equivalent distilled water control group, found that, biped is all without dead and untoward reaction during this period, and two treated animal appetite, stool and urine and body weight be there was no significant difference (p>0.05) statistically; There was no significant difference (p>0.05) between the routine blood indexes two groups such as blood leucocyte, red corpuscle and thrombocyte; Two treated animal internal organs anatomy and pathologies observe pathological phenomenon all without exception, illustrate that this fermentation mycelium meta-bolites long-term taking is safe and reliable.
(3) whole experimental result shows: You Wei can intend dish stey GMH31 fermentation mycelium meta-bolites has direct lethal effect to tumour, and has and significantly treat function of tumor; And have no side effect, safe and reliable.
Claims (7)
1. an Artemisia japonica Nei Shengyouwei can intend pestalotia bacteria strain, it is characterized in that, the outstanding Wei of called after can be intended coiling stey (Pestalotiopsis uvicola) GMH31, depositary institution: China typical culture collection center, preservation day: on June 30th, 2014, preservation registration number is CCTCCNO:M2014303.
2. outstanding Wei as claimed in claim 1 can be intended coiling stey (Pestalotiopsis uvicola) GMH31 bacterial strain, and wherein, the isolation cultivation method step of this bacterial strain is as follows:
(1) preparation of Japanese wormwood herb botanical tissue: the leaf texture gathering Artemisia japonica, with tap water, the raw microorganism of table of removing sample surfaces, is then put in wind on clean gauze and dries;
(2) sterilization and Sterility testing is shown: the tissue block after drying is cut into suitable size, rinses well with sterile purified water, blade sterilization employing 75% alcohol immersion 1-2min, 0.1%HgCl
2solution soaking 3-4min, then rinse to thimerosal noresidue with sterile purified water, after blotting tissue segments remained on surface water droplet with sterilizing filter paper, what be cut into diameter 4 ~ 6mm size organizes segment;
(3) separation and purification of endogenetic fungus: on the PDA culture medium flat plate organizing segment to be inoculated in containing Vetstrep and penicillin after step (2) being sterilized, in described PDA substratum, the concentration of Vetstrep is 100 μ gmL
-1, the concentration of penicillin is 50 μ gmL
-1, in 25-30 DEG C of constant temperature culture 2 ~ 7 days after sealing, grow from tissue block to mycelium, adopt Tip Splitting picking method, mycelia is transferred on purifying PDA culture medium flat plate, at 25-30 DEG C, carry out purifying cultivate 5 ~ 9 days, grow complete bacterium colony, be Artemisia japonica endogenetic fungus.
3. outstanding Wei as claimed in claim 2 can intend dish stey GMH31 bacterial strain, it is characterized in that: in described step (3), culture temperature is 28 DEG C, and slat chain conveyor, purifying incubation time is 7 days.
4. the outstanding Wei of control fungal diseases of plants as claimed in claim 1 can intend a preparation method of coiling stey GMH31 meta-bolites, carries out according to the following steps:
(1) activation culture of bacterial classification: the outstanding Wei being separated preservation can be intended dish stey GMH31 bacterial classification and move on potato glucose PDA solid plate, cultivate under 28 ± 1 DEG C of conditions, covering whole culture dish to mycelia, is that the punching of 7mm punch tool obtains bacterium cake, for inoculation with diameter;
(2) liquid fermentation and culture: fermentation culture is containing Semen Maydis powder 10g, N.F,USP MANNITOL 20g, glucose 20g, yeast extract paste 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 500mL triangle; Prepare rear 121 DEG C of sterilizing 20-30min, to be cooled to 40 DEG C aseptically picking 1 diameter be 7mm bacterium cake, under 28 ± 1 DEG C of conditions, standing for fermentation cultivate 28-35 days;
(3) broth extraction: collect fermented liquid after fermentation, adds the extraction into ethyl acetate of 1-3 times of volume, takes off a layer solution, again with the n-butanol extraction of 1-2 times of volume, get upper liquid after layering, be concentrated into medicinal extract under vacuum, obtain propyl carbinol fermented liquid extract.
5. preparation method as claimed in claim 4, it is characterized in that, described Plant diseases fungi is Kiwifruit brown rot germ (Sclerotinia sclerotiorum), phytophthora blight of pepper (Phytophthora capsici).
6. the outstanding Wei as claimed in claim 1 with antitumor action can intend coiling a preparation method for stey GMH31 meta-bolites, carries out according to the following steps:
(1) activation culture of bacterial classification: the outstanding Wei being separated preservation can be intended dish stey GMH31 bacterial classification and move on potato glucose PDA solid plate, cultivate under 28 DEG C ± 1 DEG C condition, covering whole culture dish to mycelia, is that the punching of 7mm punch tool obtains bacterium cake, for inoculation with diameter;
(2) liquid fermentation and culture: fermentation culture is containing Semen Maydis powder 10g, N.F,USP MANNITOL 20g, glucose 20g, yeast extract paste 5g, peptone 10g, KH in every 1000mL
2pO
40.5g, CaCO
315g, MgSO
47H
2o0.3g, the bottled fermentation culture 100mL of every 500mL triangle; Prepare rear 121 DEG C of sterilizing 20-30min, to be cooled to 40 DEG C aseptically picking 1 diameter be 7mm bacterium cake, under 28 ± 1 DEG C of conditions, standing for fermentation cultivate 28-35 days;
(3) mycelium extracts: collect mycelium after fermentation, adding concentration is 30-90% aqueous acetone solution, the concentration of mycelium in aqueous acetone solution is made to be less than or equal to 0.50g/L, ultrasonic extraction 2-5 time under power is 40KHz, the condition of 30min/ time, then under equal conditions add equal-volume ethyl acetate ultrasonic extraction 2-5 time again, filter, collect upper strata ethyl acetate solution, be concentrated into medicinal extract under vacuum, obtain ethyl acetate mycelium extract.
7. preparation method as claimed in claim 6, it is characterized in that, described tumour cell is Murine Ascitic Hepatoma Cells H22, mouse hydroperitoneum type cervical cancer cell U14 and mouse hydroperitoneum type sarcoma cell S180.
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| WO2003064617A2 (en) * | 2002-01-30 | 2003-08-07 | Montana State University | Pestalotiopsis microsporia isolates and compounds derived therefrom |
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| CN106947781A (en) * | 2017-04-21 | 2017-07-14 | 云南大学 | A kind of fungi intends disk stey extract and its application |
| CN106947781B (en) * | 2017-04-21 | 2019-08-23 | 云南大学 | A kind of fungi intends disk stey extract and its application |
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