CN1802925A - Use of gingko endogenous fungus metabolite in preventing and treating plant fungus disease - Google Patents
Use of gingko endogenous fungus metabolite in preventing and treating plant fungus disease Download PDFInfo
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- CN1802925A CN1802925A CN 200510062338 CN200510062338A CN1802925A CN 1802925 A CN1802925 A CN 1802925A CN 200510062338 CN200510062338 CN 200510062338 CN 200510062338 A CN200510062338 A CN 200510062338A CN 1802925 A CN1802925 A CN 1802925A
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Abstract
The invention discloses an application of ginko endogenetic fungi metabolite in preventing plant mycosis, belonging to microbiological pesticide technology, which comprises the following steps: preparing ginko tissue, surface sterilizing, separating and culturing the endogenetic fungi, sifting effective strain, fermenting and preparing bactericide. The invention provides a new way for developing agricultural bactericide.
Description
Technical field
The present invention relates to the microbial pesticide technical field, particularly relate to the application of gingko endogenous fungus metabolite in the control fungal diseases of plants.
Background technology
Ginkgo (Ginkgo biloba L.) has another name called gingko, is the rare famous and precious seeds in the distinctive world of China, is the Relict Plant that ice movement is left over, and is called as living fossil.Belonging to Ginkgoaceae, is the existing unique existence kind of Ginkgo.Now be extensively to cultivate seeds, have high research and development and be worth.Each tissue of ginkgo contains antibacterial substance, it is reported that the extract of ginkgo root, stem, leaf, pulp (exosper) and kernel has inhibitory action to the test plant pathogen.But the plant resources amount is one of principal element of restriction botanical fungicide development scale.According to host plant and in it living microorganism because long-term community life has " endosymbiotic theory " of same or analogous secondary metabolite route of synthesis, separation screening is to the existing report of the endogenetic fungus that produces flavones (Wang Meixia etc. from ginkgo, Nanjing Normal University's journal, 2003,26 (1)), this relates generally to the research of medical aspect.But separation screening endogenetic fungus from ginkgo, and its metabolite is applied to prevent and treat fungal diseases of plants, this yet there are no the research report at home and abroad.
Summary of the invention
The objective of the invention is from ginkgo, to separate the endogenetic fungus that metabolite has bacteriostasis, and this bacterium fermentating metabolism product is applied to prevent and treat fungal diseases of plants.
Purpose of the present invention is achieved by the following technical programs.
The application in the control fungal diseases of plants of a kind of separation screening of gingko endogenous fungus and metabolite thereof specifically may further comprise the steps:
(1) preparation of ginkgo tissue: gather ginkgo root, stem, leaf, pulp, kernel;
(2) surface sterilization of ginkgo tissue and aseptic detection; Step (1) ginkgo tissue after surface sterilization, is directly planted on PDA medium plate, put 26 ℃～28 ℃ insulating boxs and cultivate, therefrom select the tissue of the no any growth in surface;
(3) ginkgo is organized the separation and Culture of endogenetic fungus: the sterile tissue of step (2) is being cut into patch-graft on the PDA medium under the aseptic condition, putting 26 ℃～28 ℃ insulating boxs cultivates, when growing mycelia around the sample, adopt most advanced and sophisticated mycelia picking method, the bacterium colony that the picking form is different, be transferred to the PDA slant medium after purified, standby;
(4) preparation of endogenetic fungus liquid fermentation medium: its each component and every liter of contained quality are peptone 0-5.5g, corn flour 1-8g, wheat bran 0-10g, soluble starch 0-10g, glucose 1-8g, soybean cake powder 1-9g, CaCO
30.5-2.5g, NH
4NO
30.1-1.2g, MgSO
40.1-0.9g, KH
2PO
40.1-0.9g;
(5) endogenetic fungus fermented and cultured: step (4) liquid fermentation medium is divided in the triangular flask, with inoculation hook picking a little through the endogenetic fungus mycelia of step (3) separation and purification in blake bottle, put 26 ℃～28 ℃ shaking tables, 200r/min, shaken cultivation 4～5 days, zymotic fluid is got fermented supernatant fluid through the centrifugal 10min of 5000r/min;
(6) effective strain screening: get each supernatant of step (5) be cooled to 50 ℃ PDA medium by 1: 9 mixing of ml volumes in sterile petri dish, solidify the back confession examination bacterium cake that 1 diameter is 4mm of putting on each medium plane, after putting 28 ℃ of incubators cultivations, calculate each bacterial strain fermentation liquor to inhibiting rate for the examination pathogen, therefrom filter out the endogenetic fungal bacterial strain of efficient inhibiting rate, standby;
(7) gingko endogenous fungus metabolite is in the application of control in the fungal diseases of plants: the efficient bacterial strain that step (6) is filtered out, adopt step (4) liquid fermentation medium to cultivate in fermentation cylinder for fermentation, with its supernatant or fermenting mixture, be equipped with an amount of agricultural drugs auxiliary material, be prepared into the agricultural drugs of multiple formulation.
Described liquid fermentation medium, its each component and every liter of contained quality are peptone 2.5g, corn flour 3g, wheat bran 5g, glucose 4g, soluble starch 4g, soybean cake powder 4g, CaCO
31g, NH
4NO
30.6g, MgSO
40.1g, KH
2PO
40.6g;
Step of the present invention (2) and (3) described medium are PDA medium (potato glucose medium), and its component and content are: potato 200g, glucose 20g, agar 20g.Peeling potatoes is cut into piece and boils half an hour, uses filtered through gauze then, adds glucose and agar again, supplies water to 1L after dissolving.
The described phytopathogen for examination bacterium cake of step of the present invention (6) is tomato early blight bacterium (Alternariasolani), cucumber fusarium axysporum (Fusarium oxysporum), bean anthrax bacteria (Colletotrichumlindemuthianum), grape anthracnose (Colletotrichum gloeosporioides), in Rhizoctonia solani Kuhn (Thanatephorus cucumeris) or the cucumber rhizoctonia rot bacterium (Rhizoctonia solani) one or more (all being the pathogen of describing in " nosophyte mycology " in close relations) with the agricultural plant disease.
Beneficial effect of the present invention, the one, the present invention filters out its metabolite according to " endosymbiotic theory " from ginkgo and the host plant extract is same or analogous, to the inhibited endogenetic fungus of fungal diseases of plants pathogen, this provides effective strain separating screening technique for realizing by extracting the conversion of Fungicidal active substance to the Production by Microorganism Fermentation Fungicidal active substance in the plant; The 2nd, tracking and measuring of the present invention each endogenetic fungal bacterial strain metabolite of being separated to inhibitory action to the test plant pathogen, thereby obtain effective strain, for the further development and the exploitation of microbial pesticide provides a collection of starting strain; The 3rd, the present invention has paid attention to aseptic detection in the early stage of plant tissue, and has developed the liquid fermentation medium that gingko endogenous fungus is suitable for, and has improved success rate and efficient to the endogenetic fungus separation screening; The 4th, the present invention is applied to the gingko endogenous fungus fermentating metabolism product control of fungal diseases of plants first, and effect and conventional chemical medicament similar (seeing the test example) are for the exploitation of disinfectant use in agriculture has increased new approach.
Embodiment
Invention is further described to this purposes below in conjunction with embodiment.
Embodiment 1:
Gather the Caulis Ginkgo tissue from Changxing, Zhejiang ginkgo gallery, carry out surface sterilization by follow procedure: the running water flushing, 75% alcohol rinsing 5min, aseptic water washing 5 times soaked 4 minutes aseptic water washing 3 times again with mercuric chloride; Stem after the surface sterilization is organized and is directly planted on PDA medium plate, puts in 28 ℃ of insulating boxs and cultivates, and therefrom selects the stem tissue of the no any growth in surface;
To under the superclean bench condition, cut into the small pieces of about 0.5cm * 0.5cm through stem (the getting phloem) tissue of aseptic detection, then small pieces be planted on the PDA culture medium flat plate, and put 28 ℃ of insulating boxs and cultivate; When obviously growing mycelia around the sample, adopt most advanced and sophisticated mycelia picking method, the bacterium colony that the picking form is different, it is standby to be transferred to the PDA slant medium after purified;
With above-mentioned separate each bacterial strain of gingko endogenous fungus carry out liquid fermentation and culture, each component of its medium and every liter of contained quality are: peptone 2.5g, corn flour 3g, wheat bran 5g, glucose 4g, soluble starch 4g, soybean cake powder 4g, CaCO
31g, NH
4NO
30.6g, MgSO
40.1g, KH
2PO
40.6g;
The 500mL liquid fermentation medium is divided in the triangular flask of 300mL, every bottled liquid measure is 50mL; In blake bottle, place 28 ℃ of shaking tables, 200r/min, shaken cultivation 4 days with a little mycelium inoculation of inoculation hook picking; The centrifugal 10min of zymotic fluid 5000r/min gets supernatant, abandons mycelium;
The zymotic fluid bacteriostatic activity is measured: get above-mentioned each strain fermentation supernatant 1mL in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA medium with 9mL, 1 bacterium cake (diameter is 4mm) for examination bacterium cucumber rhizoctonia rot bacterium (Rhizoctonia solani) is put in the cooling back on each medium plane, the bacterium cake is connected to culture dish central authorities (the culture dish diameter is 9cm), put 28 ℃ of cultivation 36h in the incubator, handle in contrast with sterile water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:
The result shows: the metabolite that is numbered 3,64,73,121,303,431,528,555,597,820,847,920,1012,1028 bacterial strains is respectively 25%, 36%, 45%, 26%, 42%, 47%, 39%, 41.5%, 37.3%, 54%, 19.5%, 59%, 46%, 68% to the inhibiting rate of cucumber rhizoctonia rot bacterium.
Embodiment 2:
Get ginkgo leaf, press the follow procedure surface sterilization: the running water flushing, 75% alcohol rinsing 5min, aseptic water washing 5 times soaked 4 minutes aseptic water washing 3 times again with mercuric chloride; Blade after the surface sterilization is directly planted on PDA medium plate, puts in 27 ℃ of insulating boxs and cultivates, and therefrom selects the tissue of the no any growth in surface;
To under the superclean bench condition, cut into the small pieces of about 0.5cm * 0.5cm through the blade of aseptic detection, then small pieces be planted on the PDA flat board, and put 27 ℃ of insulating boxs and cultivate; When obviously growing mycelia around the sample, adopt most advanced and sophisticated mycelia picking method, the bacterium colony that the picking form is different is transferred to the PDA slant medium after purified, and is standby;
With above-mentioned separate each bacterial strain of gingko endogenous fungus carry out liquid fermentation and culture: its each component of liquid fermentation medium and every liter of contained quality are: peptone 5.5g, corn flour 1g, wheat bran 10g, glucose 8g, soybean cake powder 1g, CaCO
30.5g, NH
4NO
31.2g, MgSO
40.4g, KH
2PO
40.1g; Medium is divided in the triangular flask of 300mL, every bottled liquid measure is 50mL; In blake bottle, place 27 ℃ of shaking tables, 200r/min, shaken cultivation 5 days with a little mycelium inoculation of inoculation hook picking; The centrifugal 10min of zymotic fluid 5000r/min gets supernatant, abandons mycelium;
The zymotic fluid bacteriostatic activity is measured: get above-mentioned each bacterial strain fermentation liquor 1mL in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA medium with 9mL, 1 bacterium cake (diameter is 4mm) for examination bacterium tomato early blight bacterium (Alternaria solani) is put in the cooling back on each medium plane, the bacterium cake is connected to culture dish central authorities (the culture dish diameter is 9cm), put 28 ℃ and cultivate 72h, handle in contrast with sterile water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate, computational methods are with embodiment 1.
The result shows: the metabolite that is numbered 256,383,604,618,918 bacterial strains is respectively 56%, 38%, 67%, 20%, 42% to the inhibiting rate of tomato early blight bacterium.
Embodiment 3:
Get ginkgo pulp (exosper), press the follow procedure surface sterilization: the running water flushing, 75% alcohol rinsing 5min, aseptic water washing 5 times soaked 4 minutes aseptic water washing 3 times again with mercuric chloride; Pulp organization after the surface sterilization is directly planted on PDA medium plate, puts in 26 ℃ of insulating boxs and cultivates, and therefrom selects the fruit tissue of the no any growth in surface;
To under the superclean bench condition, cut into the small pieces of about 0.5cm * 0.5cm through the pulp of aseptic detection, then small pieces be planted on the PDA flat board, and put 26 ℃ of insulating boxs and cultivate; When obviously growing mycelia around the sample, adopt most advanced and sophisticated mycelia picking method, the bacterium colony that the picking form is different is transferred to the PDA slant medium after purified, and is standby.
With above-mentioned separate each bacterial strain of gingko endogenous fungus carry out liquid fermentation and culture: its each component of liquid fermentation medium and every liter of contained quality are: corn flour 8g, glucose 1g, soluble starch 10g, soybean cake powder 9g, CaCO
32.5g, NH
4NO
30.1g, MgSO
40.9g, KH
2PO
40.9g;
The 500mL medium is divided in the triangular flask of 300mL, every bottled liquid measure is 50mL; In blake bottle, place 26 ℃ of shaking tables, 200r/min, shaken cultivation 5 days with a little mycelium inoculation of inoculation hook picking; The centrifugal 10min of zymotic fluid 5000r/min gets supernatant, abandons mycelium;
The zymotic fluid bacteriostatic activity is measured: get above-mentioned bacterial strains zymotic fluid 1mL in sterile petri dish, be cooled to 50 ℃ the rapid mixing of PDA medium with 9mL, 1 bacterium cake (diameter is 4mm) for examination bacterium grape anthracnose (Colletotrichum gloeosporioides) is put in the cooling back on each medium plane, the bacterium cake is connected to culture dish central authorities (the culture dish diameter is 9cm), put 28 ℃ and cultivate 72h, handle in contrast with sterile water, repeat 3 times; Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate, computational methods are with embodiment 1;
The result shows: the metabolite that is numbered 78,207,305,391,634,1002 bacterial strains is respectively 25.3%, 61%, 64.6%, 43.4%, 50.9%, 61.3% to the inhibiting rate of grape anthracnose.
Embodiment 4-6 is development and the explanation of effect on the control corps diseases that the metabolite of bacterial strain of the present invention carries out the bactericide product;
Wherein 920 bacterial strain metabolites are for pressing technology and 4 days gained supernatants of formula, fermenting of embodiment 1;
604 bacterial strain metabolites are for pressing technology and 5 days gained supernatants of formula, fermenting of embodiment 2; 305 bacterial strain metabolites are for pressing technology and 5 days gained supernatants of formula, fermenting of embodiment 3;
Embodiment 4: product contains 2% 920 bacterial classification metabolites, the white carbon of adding 3% is as adsorbent, and add 1% agricultural newborn 0203-B and 3% sodium lignin sulfonate auxiliary material, supply 100% with precipitated calcium carbonate at last, behind the mixing, behind comminution by gas stream, make wetting powder (claiming " product A " in the following content).
Embodiment 5: product contains 1.5% 604 bacterial classification metabolites, dissolve with methanol with 5%, and add 2% farming breast, 601 and 0.5% dodecyl sodium sulfate as dispersing aid, add 1% urea as antifreezing agent, last water supplies 100%, 35 ℃ of following mixings are made microemulsion (claiming " product B " in the following content) after the emulsification of high-shear emulsifying device disperses.
Embodiment 6: product contains the metabolite of 3% 305 bacterial classifications, and the dissolve with ethanol with 5% adds 6% agricultural newborn 0204-C as emulsifier, supplies 100% with dimethylbenzene at last, and stirring and evenly mixing is made missible oil (claiming " products C " in the following content).
The test example:
The disinfectant use in agriculture of embodiment 4-6 trial-production is prevented and treated test to crop pests such as cucumber rhizoctonia rot, tomato early blight and bitter rot or anthracnose of grapes, with conventional chemical agricultural chemicals, clear water is contrast, as shown in table 1, the control efficiency of the disinfectant use in agriculture of trial-production and conventional chemical agricultural chemicals is similar or slightly excellent.
Table 1 product A, B, C are to several crop pest preventive effect statistical forms
(test of pesticide effectiveness is all carried out in test farmland, suburbs, Hangzhou in May, 2004-August)
| Product and contrast medicine | Extension rate | Using method | Proofread and correct preventive effect (%) | ||
| Cucumber rhizoctonia rot | Tomato early blight | Bitter rot or anthracnose of grape | |||
| Product A | 1000 | Spraying | 92.3 | - | - |
| 1500 | Spraying | 88.5 | - | - | |
| 2000 | Spraying | 80.4 | - | - | |
| But 70% thiophanate methyl wet-milling | 1000 | Spraying | 89.7 | - | - |
| Product B | 800 | Spraying | - | 93.2 | - |
| 1200 | Spraying | - | 86.5 | - | |
| 1600 | Spraying | - | 81.7 | - | |
| But 50% carbendazim wet-milling | 500 | Spraying | - | 84.5 | - |
| Products C | 1000 | Spraying | - | - | 94.8 |
| 1500 | Spraying | - | - | 90.5 | |
| 2000 | Spraying | - | - | 83.8 | |
| But 70% mancozeb wet-milling | 800 | Spraying | - | - | 91.5 |
| Clear water | - | Spraying | 4.3 | -10.2 | 2.8 |
"-" is not test (N.T.).
Claims (2)
1, the application of gingko endogenous fungus metabolite in the control fungal diseases of plants.
2, the application in the control fungal diseases of plants according to claim 1 is characterized in that: after strain fermentation is cultivated, with its supernatant or fermenting mixture, be equipped with the agricultural drugs auxiliary material, be prepared into the agricultural drugs of multiple formulation.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100623380A CN1315387C (en) | 2005-12-30 | 2005-12-30 | Use of gingko endogenous fungus metabolite in preventing and treating plant fungus disease |
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|---|---|---|---|
| CNB2005100623380A CN1315387C (en) | 2005-12-30 | 2005-12-30 | Use of gingko endogenous fungus metabolite in preventing and treating plant fungus disease |
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| CN1802925A true CN1802925A (en) | 2006-07-19 |
| CN1315387C CN1315387C (en) | 2007-05-16 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110558336A (en) * | 2019-07-19 | 2019-12-13 | 湖南科技学院 | Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof |
| WO2022188162A1 (en) * | 2021-03-12 | 2022-09-15 | 德州学院 | Endophytic fungus in ginkgo biloba and anti-bacterial and antitumor application thereof |
-
2005
- 2005-12-30 CN CNB2005100623380A patent/CN1315387C/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110558336A (en) * | 2019-07-19 | 2019-12-13 | 湖南科技学院 | Biocontrol agent for preventing and treating lettuce sclerotinia rot and preparation and using method thereof |
| WO2022188162A1 (en) * | 2021-03-12 | 2022-09-15 | 德州学院 | Endophytic fungus in ginkgo biloba and anti-bacterial and antitumor application thereof |
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| Publication number | Publication date |
|---|---|
| CN1315387C (en) | 2007-05-16 |
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Granted publication date: 20070516 Termination date: 20111230 |