CN103194490A - Method for preparing phytopathogen-resisting ginkgo biloba L. endophytic-fungus fermentation broth - Google Patents
Method for preparing phytopathogen-resisting ginkgo biloba L. endophytic-fungus fermentation broth Download PDFInfo
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Abstract
The invention relates to a method for preparing a phytopathogen-resisting ginkgo biloba L. endophytic-fungus fermentation broth. The ginkgo biloba L. endophytic fungus is fusarium solani T-7 which is collected in the CGMCC (China General Microbiological Culture Collection Center) on July 20, 2011 with the collection number of CGMCC No. 5089. The method comprises the following steps of: (1) activating bacteria; (2) carrying out primary seed culturing and secondary seed culturing; and (3) culturing by fermenting. The antibacterial experiments find that the fusarium solani T-7 antibacterial fermentation broth has significant effects of inhibiting the growth of 5 plant pathogenic fungi including the tomato oxysporum, the apple cytospora mandshurica, the apple colletotrichum gloeosporioides, the pear venturia inaequalis and the wheat fusarium graminearum. A novel agricultural antibiotic natural active substance for resisting the plant pathogenic fungus disease is prepared by taking a plant endophytic fungus as the resource.
Description
Technical field
The invention belongs to microbial technology field, relate to a kind of fermentation culture method, be specifically related to the antibiotic preparation of fermentation liquid method of gingko endogenous fungus.
Background technology
In the agricultural production process, use chemical pesticide to cause that a series of environment and health problem are extensively cognitive by the people in a large number, development safety, novel agrochemical efficient, environmental protection have become the direction of development and the theme of research.Microbial pesticide is difficult for producing resistance because of environmentally friendly, and production cost is low, and zymotechnique is simple, and non-target organism is waited advantage safely, becomes the focus and emphasis of novel agrochemical development.As the plant endogenesis epiphyte (Endophytic fungi) in one of important medicine source, interacting for a long time with host plant, in the process of coevolution, in plant materials, keeping the microecosystem balance.In order to keep this balance, plant endogenesis epiphyte may produce the secondary metabolite with the same or similar function of host plant.Therefore, can be with the secondary metabolite of the plant endogenesis epiphyte potential or alternate resources as new compound, novel drugs screening, thus effectively reduce the R﹠D costs of novel drugs or other purposes active substance.
Ginkgo (
Ginkgo bilobaL.) have the title of gymnosperm " living fossil " and " vegitabilia panda ", is Chinese distinctive rare tree, infects disease and pest in very long vegetative period hardly, and the life-span is extremely long.Infer according to the plant endogenesis epiphyte endosymbiotic theory, probably exist some endogenetic fungus in the ginkgo, have the secondary metabolite of special anti-microbial activity by generation, influence the disease resistance ability of host ginkgo.
Summary of the invention
The object of the present invention is to provide a kind of mycelial growth to plant pathogenic fungi to have the antibiotic preparation of fermentation liquid method of better inhibiting gingko endogenous fungus.
Gingko endogenous fungus called after fusarium solanae T-7 of the present invention (
Fusarium solaniT-7), now be deposited in the China Committee for Culture Collection of Microorganisms common micro-organisms center of State Intellectual Property Office's appointment, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on July 20th, 2011, deposit number is CGMCC No.5089.
Gingko endogenous fungus of the present invention be therefrom state Hefei City, Anhui Province ginkgo (
Ginkgo bilobaL) separation obtains in the plant living body.
The concrete preparation method of antibiotic fermented liquid is as follows:
The concrete preparation manipulation step of described antibiotic fermented liquid is as follows:
(1) actication of culture
Under aseptic condition, use the bacterial classification of the gingko endogenous fungus of a little preservation of transfering loop picking, to transfer in sterilized potato dextrose agar solid medium flat board, the constant temperature lucifuge was cultivated 5 days, and 28 ℃ ± 1 ℃ of culture temperature obtains activated spawn;
(2) seed culture
Under the aseptic condition, utilize transfering loop picking 3 ring activated spawn, be inoculated in the potato glucose liquid nutrient medium of 50ml, the constant temperature shaking table was cultivated 2 days, 160 rev/mins of shaking speed, and 28 ℃ ± 1 ℃ of temperature obtains first order seed; With first order seed, with 10% inoculum size, be inoculated in the liquid nutrient medium of 50ml, the constant temperature shaking table was cultivated 2 days, 160 rev/mins of shaking speed, 28 ℃ ± 1 ℃ of temperature obtains secondary seed;
(3) fermentation culture
Secondary seed is inoculated in the liquid nutrient medium of 50ml, inoculum size 10%, the constant temperature shaking table was cultivated 7 days, 160 rev/mins of shaking speed, 28 ℃ ± 1 ℃ of temperature obtains the gingko endogenous fungus fermented liquid; Filtering fermentation liquor is removed mycelium, under the aseptic condition, crosses 0.22 micron filter, namely obtains the antibiotic fermented liquid of gingko endogenous fungus T-7.
The fermentation culture feature: cultivated the 1st day, thalline is the powder of a small amount of pale pink; Cultivated the 3rd day, the fine powder of pale pink reduces, and the little mycelium pellet of pink colour occurs; Cultivated the 5th day, pink mycelium pellet continues to increase, and it is big that volume becomes, and fermented liquid is orange red; Cultivated the 7th day, and a large amount of red mycelium pellets occurred, it is big that volume becomes, and fermented liquid is orange red.
Described potato dextrose agar solid medium: 1 liter of potato 200 gram, glucose 20 grams, agar 18 grams, distilled water.
The preparation of described potato glucose liquid nutrient medium: potato 200 grams, 1 liter of glucose 20 grams, distilled water, pH nature.
Described liquid nutrient medium: peptone 15 grams, glucose 20 grams, SODIUMNITRATE 1 gram, calcium chloride 0.5 gram, sal epsom 0.1 gram, dipotassium hydrogen phosphate 0.1 gram, 1 liter of distilled water, pH nature.
Under 121 ℃ of temperature, pressure 0.1MPa, 30 min sterilized before above-mentioned three kinds of substratum used.
The antibacterial tests of the antibiotic fermented liquid of the present invention by gingko endogenous fungus T-7 finds, this bacterium to for examination 5 kind of plant pathogenic fungies (the tomato wilt bacterium (
Fusarium oxysporum), Valsa mali
(Cytospora mandshurica),Apple anthrax bacteria
(Colletotrichum gloeosporioides),Pear cucumerinum (
Venturia pirina)
,Fusarium graminearum (
Fusarium graminearum)) mycelial growth have comparatively obvious suppression effect.The present invention with plant endogenesis epiphyte as resource, in the hope of obtaining the novel agricultural antibiotics natural radioactivity thing of anti-plant pathogenic fungi disease.
Embodiment
The antibacterial tests of the anti-pathogenic fermented liquid of gingko endogenous fungus T-7 endogenetic fungus of the present invention is as follows:
1. plant pathogenic fungi:
The Phytophthora capsici germ (
Phytophthora capsici), the tomato wilt bacterium (
Fusarium oxysporum), Valsa mali (
Cytospora mandshurica), apple anthrax bacteria (
Colletotrichum gloeosporioides), pear cucumerinum (
Venturia pirina), fusarium graminearum (
Fusarium graminearum);
2. the cultivation of pathogenic micro-organism
The slant culture of getting plant pathogenic fungi inserts the dull and stereotyped activation of potato dextrose agar solid medium respectively, cultivates 5 days in (28 ± 1) ℃ thermostat container.
3. the cultivation of endogenetic fungus:
The used gingko endogenous fungus called after of present embodiment fusarium solanae T-7 (
Fusarium solaniT-7), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, the address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, preservation date on July 20th, 2011, deposit number is CGMCC No.5089;
Under aseptic condition, with the bacterial classification of a little gingko endogenous fungus of transfering loop picking, to transfer in sterilized potato dextrose agar solid medium flat board, the constant temperature lucifuge was cultivated 5 days, and 28 ℃ ± 1 ℃ of culture temperature obtains activated spawn.
4.T-7 bacterial strain fermentation liquor preparation
Under the aseptic condition, utilize transfering loop picking 3 ring activated spawn, be inoculated in the potato glucose liquid nutrient medium of 50ml, the constant temperature shaking table was cultivated 2 days, 160 rev/mins of shaking speed, and 28 ℃ ± 1 ℃ of temperature obtains first order seed; With first order seed, with 10% inoculum size, be inoculated in the liquid nutrient medium of 50ml, the constant temperature shaking table was cultivated 2 days, 160 rev/mins of shaking speed, 28 ℃ ± 1 ℃ of temperature obtains secondary seed;
Secondary seed is inoculated in the liquid nutrient medium of 50ml, inoculum size 10%, the constant temperature shaking table was cultivated 7 days, 160 rev/mins of shaking speed, 28 ℃ ± 1 ℃ of temperature obtains the gingko endogenous fungus fermented liquid; Filtering fermentation liquor is removed mycelium, under the aseptic condition, crosses 0.22 micron filter, namely obtains the antibiotic fermented liquid of gingko endogenous fungus T-7;
The fermentation culture feature: cultivated the 1st day, thalline is the powder of a small amount of pale pink; Cultivated the 3rd day, the fine powder of pale pink reduces, and the little mycelium pellet of pink colour occurs; Cultivated the 5th day, pink mycelium pellet continues to increase, and it is big that volume becomes, and fermented liquid is orange red; Cultivated the 7th day, and a large amount of red mycelium pellets occurred, it is big that volume becomes, and the antibiotic fermented liquid of gingko endogenous fungus T-7 is orange red;
The preparation of above-mentioned potato glucose liquid nutrient medium: potato 200 grams, 1 liter of glucose 20 grams, distilled water, pH nature;
Above-mentioned potato dextrose agar solid medium: 1 liter of potato 200 gram, glucose 20 grams, agar 18 grams, distilled water;
Aforesaid liquid substratum: peptone 15 grams, glucose 20 grams, SODIUMNITRATE 1 gram, calcium chloride 0.5 gram, sal epsom 0.1 gram, dipotassium hydrogen phosphate 0.1 gram, 1 liter of distilled water, pH nature;
Under 121 ℃ of temperature, pressure 0.1MPa, 30 min sterilized before above-mentioned three kinds of substratum used.
5. suppress the mycelial growth rate method and measure anti-microbial activity
Under the aseptic condition, in 100 milliliters of triangular flasks, add the antibiotic fermented liquid of gingko endogenous fungus of 6 milliliters of the present invention's preparations and the substratum of 54 milliliters of aseptic thawings of potato dextrose agar, shake up, getting 10 milliliters respectively, to place diameter be to make flat board in 9 centimetres of sterile petri dish, insert 1 after cooling on each substratum plane for examination pathogenic bacteria bacterium cake (5 millimeters of diameters), the mycelia face of bacterium cake is attached to media surface, places 28 ± 1 ℃ of lucifuges to cultivate 72 hours flat board.Adopt the right-angled intersection method to measure the colony growth diameter, calculate inhibiting rate with following formula:
Table 1: bacterial strain T-7 fermented product of the present invention is to the The anti-bacterial result of six kind of plant pathogenic micro-organisms
By table 1 as seen, fusarium solanae T-7 fermenation raw liquid is to Valsa mali
(Cytospora mandshurica)Mycelial growth inhibition rate reaches more than 85%, to pear cucumerinum (
Venturia pirina) mycelial growth inhibition rate more than 35%, to fusarium graminearum (
Fusarium graminearum), the tomato wilt bacterium (
Fusarium oxysporum), apple anthrax bacteria
(Colletotrichum gloeosporioides)Mycelial growth also have certain restraining effect.This shows, the present invention prepares resulting gingko endogenous fungus endogenetic fungus fusarium solanae T-7 fermented product the test plant pathogenic fungi is had tangible anti-microbial activity, it can be elected to be the endogenetic fungus resource, by fermentative preparation, in the hope of obtaining the natural component that possesses the novel agricultural activity of anti-plant pathogenic fungi disease.
Claims (1)
1. have the antibiotic preparation of fermentation liquid method of gingko endogenous fungus of anti-phytopathogen effect, it is characterized in that: described gingko endogenous fungus called after fusarium solanae T-7 (
Fusarium solaniT-7), be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center, preservation date on July 20th, 2011, deposit number is CGMCC No.5089;
The concrete preparation manipulation step of described antibiotic fermented liquid is as follows:
(1) actication of culture
Under aseptic condition, use the bacterial classification of the gingko endogenous fungus of a little preservation of transfering loop picking, to transfer in sterilized potato dextrose agar solid medium flat board, the constant temperature lucifuge was cultivated 5 days, and 28 ℃ ± 1 ℃ of culture temperature obtains activated spawn;
(2) seed culture
Under the aseptic condition, utilize transfering loop picking 3 ring activated spawn, be inoculated in the potato glucose liquid nutrient medium of 50ml, the constant temperature shaking table was cultivated 2 days, 160 rev/mins of shaking speed, and 28 ℃ ± 1 ℃ of temperature obtains first order seed; With first order seed, with 10% inoculum size, be inoculated in the liquid nutrient medium of 50ml, the constant temperature shaking table was cultivated 2 days, 160 rev/mins of shaking speed, 28 ℃ ± 1 ℃ of temperature obtains secondary seed;
(3) fermentation culture
Secondary seed is inoculated in the liquid nutrient medium of 50ml, inoculum size 10%, the constant temperature shaking table was cultivated 7 days, 160 rev/mins of shaking speed, 28 ℃ ± 1 ℃ of temperature obtains the gingko endogenous fungus fermented liquid; Filtering fermentation liquor is removed mycelium, under the aseptic condition, crosses 0.22 micron filter, namely obtains the antibiotic fermented liquid of gingko endogenous fungus T-7;
The fermentation culture feature: cultivated the 1st day, thalline is the powder of a small amount of pale pink; Cultivated the 3rd day, the fine powder of pale pink reduces, and the little mycelium pellet of pink colour occurs; Cultivated the 5th day, pink mycelium pellet continues to increase, and it is big that volume becomes, and fermented liquid is orange red; Cultivated the 7th day, and a large amount of red mycelium pellets occurred, it is big that volume becomes, and the antibiotic fermented liquid of gingko endogenous fungus T-7 is orange red;
Described potato dextrose agar solid medium: 1 liter of potato 200 gram, glucose 20 grams, agar 18 grams, distilled water;
The preparation of described potato glucose liquid nutrient medium: potato 200 grams, 1 liter of glucose 20 grams, distilled water, pH nature;
Described liquid nutrient medium: peptone 15 grams, glucose 20 grams, SODIUMNITRATE 1 gram, calcium chloride 0.5 gram, sal epsom 0.1 gram, dipotassium hydrogen phosphate 0.1 gram, 1 liter of distilled water, pH nature;
Under 121 ℃ of temperature, pressure 0.1MPa, 30 min sterilized before above-mentioned three kinds of substratum used.
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Cited By (5)
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| CN106867542A (en) * | 2015-12-14 | 2017-06-20 | 灵武市森保科技开发有限公司 | A kind of facilities vegetable continuous cropping soil conditioner and preparation method thereof |
| CN108624527A (en) * | 2018-05-12 | 2018-10-09 | 湖南科技学院 | A kind of ginkgo source growth-promoting preparation of prevention ginger bacterial wilt |
| CN109082445A (en) * | 2018-08-30 | 2018-12-25 | 德州学院 | The metabolite product of one plant of gingko endogenous fungus and its application in antibacterial |
| CN111662830A (en) * | 2020-07-14 | 2020-09-15 | 哈尔滨学院 | Entomopathogenic fungi for inhibiting wheat damping-off and preparation method thereof |
| CN114717119A (en) * | 2022-02-23 | 2022-07-08 | 广西师范大学 | Sarcandra glabra endophytic fungus and application thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106867542A (en) * | 2015-12-14 | 2017-06-20 | 灵武市森保科技开发有限公司 | A kind of facilities vegetable continuous cropping soil conditioner and preparation method thereof |
| CN106867542B (en) * | 2015-12-14 | 2020-04-21 | 灵武市森保科技开发有限公司 | Soil conditioner for facility vegetable continuous cropping and preparation method thereof |
| CN108624527A (en) * | 2018-05-12 | 2018-10-09 | 湖南科技学院 | A kind of ginkgo source growth-promoting preparation of prevention ginger bacterial wilt |
| CN109082445A (en) * | 2018-08-30 | 2018-12-25 | 德州学院 | The metabolite product of one plant of gingko endogenous fungus and its application in antibacterial |
| CN109082445B (en) * | 2018-08-30 | 2020-06-05 | 德州学院 | Metabolite product of ginkgo endophytic fungi and application of metabolite product in antibiosis |
| CN111662830A (en) * | 2020-07-14 | 2020-09-15 | 哈尔滨学院 | Entomopathogenic fungi for inhibiting wheat damping-off and preparation method thereof |
| CN114717119A (en) * | 2022-02-23 | 2022-07-08 | 广西师范大学 | Sarcandra glabra endophytic fungus and application thereof |
| CN114717119B (en) * | 2022-02-23 | 2023-06-02 | 广西师范大学 | Sarcandra glabra endophytic fungus and application thereof |
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Application publication date: 20130710 |