CN102204568A - Agricultural biological activity and application of metabolite of Platycladus orientalis endophytic fungus - Google Patents
Agricultural biological activity and application of metabolite of Platycladus orientalis endophytic fungus Download PDFInfo
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- CN102204568A CN102204568A CN2010101549802A CN201010154980A CN102204568A CN 102204568 A CN102204568 A CN 102204568A CN 2010101549802 A CN2010101549802 A CN 2010101549802A CN 201010154980 A CN201010154980 A CN 201010154980A CN 102204568 A CN102204568 A CN 102204568A
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Abstract
The invention (the title is agricultural biological activity and application of metabolite of Platycladus orientalis endophytic fungus) relates to a Platycladus orientalis endophytic fungus, a separation cultivation method thereof, and an inhibition effect of a metabolite of the fungus on main agricultural pests and weeds. The fungus, which is separated from Platycladus orientalis, has a good bacteriostatic activity on various agricultural plant pathogenic fungi such as Valsa mali, Elsinoe ampelina, Verticillium dahliae, Fusarium oxysporum and the like, and has a certain inhibition effect on various weeds such as Amaranthus retroflexus, wild oat and the like and various pests.
Description
Technical field
The invention belongs to the microbial pesticide technical field, particularly relate to the agricultural biological activity and the application thereof of arbor-vitae endogenous fungus metabolite.
Background technology
Arbor-vitae [Platycladus orientalis (L.) Franco] belongs to Cupressaceae (Cupressaceae) Platycladus (Platycladus Spach.), is China's endemic tree, is one of the especially northern urban afforestation in city seeds commonly used.Cacumen biotae gas delicate fragrance, bitter and puckery flavor, little suffering have cooling blood and hemostasis, removing heat from the lung to relieve cough effect, and planting benevolence can be used as medicine, energy mental-tranquilization, hidroschesis ease constipation.Cacumen Platycladi extract has bacteriostatic activity in various degree to golden staphylococci and Escherichia coli, and its rhizosphere soil extract has inhibitory action to the sprouting and the growth of seedling of plant seeds such as radish.
Qingdao Agricultural University is from the pesticide activity of the arbor-vitae that begins one's study in 2005, find each opzyme of arbor-vitae to apple decay, grape white rot, Pu Tao ?phytopathogen such as acne strong inhibitory action is all arranged, wherein the bacteriostatic activity of leaf extract is best, and further from leaf, being separated to bacteriostatic compound 8-methylcoumarin, national inventing patent has been applied in correlative study.Yet well-known, plant resources is one of principal element of restriction botanical pesticide large-scale production and use.The applicant is according to host plant and the long-term coevolution of living microorganism in it, and have " endosymbiotic theory " of same or similar secondary metabolite biosynthesis pathway, separation screening is to there being remarkable inhibiting endogenetic fungus to the various agricultural disease pest and weed from arbor-vitae, and bibliographical information is not seen in correlative study.
Summary of the invention
The objective of the invention is from arbor-vitae, to separate the endogenetic fungus that metabolite has pesticide activity, and this bacterium fermentating metabolism product is applied to agricultural upward to prevent and treat disease, insect pest and the crop smothering of crop.
Purpose of the present invention is achieved by the following technical programs:
The application in agriculture disease pest and weed control of the separation screening of arbor-vitae endogenetic fungus and metabolite thereof specifically may further comprise the steps:
(1) preparation of arbor-vitae tissue: root, stem, the leaf of gathering healthy arbor-vitae.
(2) surface sterilization of arbor-vitae tissue and aseptic detection: step (1) arbor-vitae tissue after surface sterilization, directly is inoculated in the PDA media surface, places (27 ± 1) ℃ insulating box to cultivate, therefrom select the tissue of the no any growth in surface.
(3) arbor-vitae 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, place (27 ± 1) ℃ insulating box to cultivate, 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) configuration of endogenetic fungus liquid fermentation medium: every liter contains 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
40.1-0.9 g, KH
2PO
40.1-0.9g all the other are sterile water.
(5) endogenetic fungus fermented and cultured: be contained in step (4) liquid fermentation medium in the triangular flask respectively, with inoculation hook picking a little through the endogenetic fungus mycelia of step (3) separation and purification in blake bottle, place (27 ± 1) ℃ shaking table, 200r/min, shaken cultivation 4-5 days, zymotic fluid is centrifugal through 5000r/min, gets fermented supernatant fluid.
(6) effective strain screening: get each supernatant of step (5) and be mixed in the sterile petri dish in by volume 1: 9 with the PDA medium that is cooled to 50 ℃, solidify the back confession examination bacterium cake that a diameter is 4mm of on each culture medium flat plate, putting, putting 28 ℃ of incubators cultivates, calculate each bacterial strain fermentation liquor to inhibiting rate for the examination pathogen, therefrom filter out efficient antibacterial endogenetic fungal bacterial strain, standby.
(7) application of arbor-vitae endogenous fungus metabolite in control plant disease pest and weed: 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.
The present invention (6) is described to be Valsa mali (Valsa mali), anthracnose of grape bacterium (Elsinoe ampelina), verticillium dahliae (Verticillium dahliae), withered germ of water-melon (Fusarium oxysporum) for the examination bacterium.
Embodiment
Invention is further described to this purposes below in conjunction with embodiment.
Embodiment 1: the typical separate of arbor-vitae endogenetic fungus is cultivated
Gather tissues such as arbor-vitae cauline leaf from Qingdao Agricultural University campus, running water is cleaned and is dried, 75% alcohol immersion 5min, and 0.1% mercuric chloride soaks 2min, carries out surface sterilization, uses rinsed with sterile water at last 5 times.Standby with the aseptic filter paper suck dry moisture.Stem tissue after the surface sterilization directly is inoculated in the PDA media surface, places (27 ± 1) ℃ insulating box to cultivate, and therefrom selects the stem tissue of the no any growth in surface.
Under germ-free condition, above-mentioned aseptic stem tissue is cut into the fragment of 0.2cm * 0.2cm, insert in the fresh PDA medium, place the constant incubator of (27 ± 1) ℃ to cultivate.When obviously growing mycelia around the sample, adopt the most advanced and sophisticated picking method of mycelia, according to the mycelia color, form and the sequencing that grows, the picking mycelia is transferred on the new PDA medium and cultivates, and purifying is until obtaining single bacterial strain repeatedly, and it is standby that numbering is preserved on the inclined-plane.
With above-mentioned separate each bacterial strain of arbor-vitae endogenetic fungus carry out liquid fermentation and culture, each component of medium and every liter of contained quality are peptone 2.5g, corn flour 3g, wheat bran 5g, soluble starch 4g, glucose 4g, soybean cake powder 4g, CaCO
31g, NH
4NO
30.6g, MgSO
40.g, KH
2PO
40.6g;
The 500mL liquid fermentation medium is contained in respectively in the 300mL triangular flask, and every bottled liquid measure is 50mL; In blake bottle, place (27 ± 1) ℃ shaking table with a little mycelium inoculation of inoculation hook picking, 200r/min, shaken cultivation 5 days, zymotic fluid is got fermented supernatant fluid through the centrifugal 10min of 5000r/min.
Embodiment 2: arbor-vitae endophyte typical activity (sterilization) screening
Get above-mentioned each strain fermentation supernatant 1mL in sterile petri dish, with the cold rapid mixing of PDA medium that goes to 50 ℃ of 9mL, putting a diameter after the cooled and solidified on each culture medium flat plate is the confession examination bacterium cake of 4mm, puts 28 ℃ of incubators and cultivates.Handle in contrast triplicate with sterile water.Adopt the right-angled intersection method to measure colony diameter, calculate inhibiting rate with following formula:
Mycelial growth inhibition rate=(contrast colony diameter-processing colony diameter) * 100%/(contrast colony diameter-4)
The result shows: have 21 strains at least 1 plant species disease fungus to be had antagonism (account for total separated strain number 19.1%), the endogenetic fungus that is numbered G21, J77, J86, Y51, Y57, Y127 has stronger antagonism to Valsa mali and anthracnose of grape bacterium.
Embodiment 3: the preparation of arbor-vitae endophyte exemplary formulations
Product contains 2% J77 bacterial classification metabolite, adds 3% white carbon as adsorbent, and adds 1% agricultural newborn 0203-B and 3% sodium lignin sulfonate auxiliary material, supplies 100% with precipitated calcium carbonate at last, behind the mixing, makes wetting powder through comminution by gas stream.
Claims (2)
1. the pesticide activity of arbor-vitae endogenous fungus metabolite and the application of disease, worm, crop smothering on the control crop thereof
2. according to claim 1 in the application that prevents and treats disease, worm, crop smothering on the crop, it is characterized in that: after strain fermentation is cultivated,, be equipped with the agricultural drugs auxiliary material, be prepared into the agricultural drugs of multiple formulation with its supernatant or fermenting mixture.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2010101549802A CN102204568A (en) | 2010-03-31 | 2010-03-31 | Agricultural biological activity and application of metabolite of Platycladus orientalis endophytic fungus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101549802A CN102204568A (en) | 2010-03-31 | 2010-03-31 | Agricultural biological activity and application of metabolite of Platycladus orientalis endophytic fungus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642704A (en) * | 2013-12-16 | 2014-03-19 | 中国农业科学院棉花研究所 | Cotton endophytic fungi CEF-714 and application of cotton endophytic fungi CEF-714 to preventing and treating cotton verticillium wilt |
| CN103834580A (en) * | 2014-03-12 | 2014-06-04 | 中国农业科学院棉花研究所 | Endophytic fungi CEF-642 of cotton and application thereof |
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| RU1793837C (en) * | 1990-11-30 | 1993-02-07 | Малое Предприятие "Агростим" | Composition for pre-sowing treatment of seeds |
| CN1720909A (en) * | 2003-11-08 | 2006-01-18 | 厦门大学 | Application of Cytospora bacterin B in preparation of antifundal medicine |
| CN101126070A (en) * | 2007-05-28 | 2008-02-20 | 中国药科大学 | Fermentation liquor formulation and condition for inducing plant endogenesis epiphyte generating activity |
| CN101225430A (en) * | 2008-02-14 | 2008-07-23 | 江苏科技大学 | Separation screening method for antibiotic antituberculotic plant endophyte |
| CN101235355A (en) * | 2008-03-03 | 2008-08-06 | 中国热带农业科学院环境与植物保护研究所 | Plants endogenetic fungus and application thereof |
| WO2009061950A2 (en) * | 2007-11-09 | 2009-05-14 | Montana State University | Endophytic fungi from pteromischum sp. plant, compounds and methods of use |
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2010
- 2010-03-31 CN CN2010101549802A patent/CN102204568A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU1793837C (en) * | 1990-11-30 | 1993-02-07 | Малое Предприятие "Агростим" | Composition for pre-sowing treatment of seeds |
| CN1720909A (en) * | 2003-11-08 | 2006-01-18 | 厦门大学 | Application of Cytospora bacterin B in preparation of antifundal medicine |
| CN101126070A (en) * | 2007-05-28 | 2008-02-20 | 中国药科大学 | Fermentation liquor formulation and condition for inducing plant endogenesis epiphyte generating activity |
| WO2009061950A2 (en) * | 2007-11-09 | 2009-05-14 | Montana State University | Endophytic fungi from pteromischum sp. plant, compounds and methods of use |
| CN101225430A (en) * | 2008-02-14 | 2008-07-23 | 江苏科技大学 | Separation screening method for antibiotic antituberculotic plant endophyte |
| CN101235355A (en) * | 2008-03-03 | 2008-08-06 | 中国热带农业科学院环境与植物保护研究所 | Plants endogenetic fungus and application thereof |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103642704A (en) * | 2013-12-16 | 2014-03-19 | 中国农业科学院棉花研究所 | Cotton endophytic fungi CEF-714 and application of cotton endophytic fungi CEF-714 to preventing and treating cotton verticillium wilt |
| CN103642704B (en) * | 2013-12-16 | 2016-05-04 | 中国农业科学院棉花研究所 | Cotton endogenetic fungus CEF-714 and the application in cotton verticillium wilt control thereof |
| CN103834580A (en) * | 2014-03-12 | 2014-06-04 | 中国农业科学院棉花研究所 | Endophytic fungi CEF-642 of cotton and application thereof |
| CN103834580B (en) * | 2014-03-12 | 2016-02-03 | 中国农业科学院棉花研究所 | The endogenetic fungus CEF-642 of cotton and application thereof |
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Application publication date: 20111005 |