CN103014068B - Active matter and application thereof - Google Patents
Active matter and application thereof Download PDFInfo
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- CN103014068B CN103014068B CN 201210580357 CN201210580357A CN103014068B CN 103014068 B CN103014068 B CN 103014068B CN 201210580357 CN201210580357 CN 201210580357 CN 201210580357 A CN201210580357 A CN 201210580357A CN 103014068 B CN103014068 B CN 103014068B
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Abstract
The invention relates to an active matter and an application thereof, which belongs to the technical field of biological pesticide. The active matter is obtained through the following steps: a, storing proteus sp. CD-126 with the preservation number of CGMCC No. 6357 in an LB culture medium of yeast extract and tryptone, wherein the culture medium comprises 5.0g of the yeast extract, 10.0g of the tryptone, 10.0g of sodium chloride and 1000ml of water, and pH of the culture medium is 7.0; activating for 24 hours in the same liquid culture medium for use; b, inoculating 100ul of the activated stain into a triangular flask containing 50-250ml of the liquid LB culture medium, and culturing for 96 hours under a condition with the temperature at 28-37 DEG C and the rotation speed at 120-180rpm to obtain liquid broth; and centrifuging the culture broth for 10 minutes at 8500rpm, and then taking the supernatant to place in a refrigerator at 4 DEG C for later use. The invention also relates to the application of the active matter in preparing biological reagents for killing M. incognita. The active matter provided by the invention has the advantages of low cost, simple preparation, high efficiency and low toxicity, and the active matter is applicable to the preparation of the biological reagents for killing the M. incognita.
Description
Technical field:
The present invention relates to a kind of actives and application thereof, belong to biological pesticide technical field.
Background technology:
Root knot nematode is a kind of plant nematode of extremely difficult control.Along with the fast development of Installation Vegetable Cultivation, the generation area of root knot nematode constantly enlarges, and harm increasingly sharpens.Root knot nematode has more than 90 to plant, and wherein Meloidogyne incognita (Meloidogyne incognita) endangers one of the most serious root knot nematode.Meloidogyne incognita (M. incognita) belongs to Nematoda (Nematoda), side tail gland mouth guiding principle (Secernentea), Tylenchida (Tylenchida), pad sword suborder (Tylenchina), Tylenchoidea (Tylenchoidea), golden nematode section (Heteroderidae), root knot subfamily (Meloidogyninae), Meloidogyne (Meloidogyne).Meloidogyne incognita (M. incognita) host range is wide, plant root after being injured forms the warty root that differs in size, and moisture and nutriment transportation are obstructed, and cause plant nutrition bad, the whole strain of falling ill when serious is wilted withered, is a kind of soil-borne disease of extremely difficult control.
The sick method of preventing and treating of existing Meloidogyne incognita (M. incognita) comprises chemical prevention, breeding for disease resistance, physical control etc., wherein take chemical nematocides as main.Along with the pollution that chemical agent causes environment, some effective nematocidess are progressively disabled, so people make every effort to seek the natural medicament that can prevent and treat Meloidogyne incognita (M. incognita) disease from occurring in nature.Microorganism is one of main source that produces bioactive natural product, and seeking to have from the meta-bolites of microorganism has the natural product of cytotoxicity to become a study hotspot to Meloidogyne incognita (M. incognita).
Summary of the invention:
The object of the present invention is to provide a kind of actives and application thereof.
The present invention is achieved in that
1, the preparation of active-fermented broth:
The bacterial isolates that the present invention uses was Bacillus proteus (Proteus sp.) CD-126, and bacterial strain CD-126 has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on July 12nd, 2012; Depositary institution address: No. 3 Institute of Microorganism, Academia Sinica in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City; Preserving number: CGMCC No.6357.
Bacillus proteus (Proteus sp.) CD-126 is kept at yeast extract Tryptones (LB) substratum (yeast extract: 5.0 g, Tryptones: 10.0 g, sodium-chlor: 10.0 g, water 1000 ml, pH:7.0) in, in the liquid nutrient medium of LB, activate 24 hours during use.Bacterial classification 100 μ l are inoculated in and are equipped with in the 50 ml-250 ml liquid LB substratum triangular flask after will activating, and are 28-37 ℃ in temperature, and rotating speed is to cultivate 96 hours under the condition of 120-180rpm, namely obtains nutrient solution.With nutrient solution 8500 rpm after centrifugal 10 minutes, getting supernatant, to put into 4 ℃ refrigerator for subsequent use, obtains the active-fermented broth that the present invention uses.
The Meloidogyne incognita (M. incognita) of 2 preparation test usefulness
Tomato Root System is used for the preparation of Meloidogyne incognita (M. incognita).Get the serious Tomato Root System of morbidity, statistics root knot quantity is put into the water soaked overnight after cleaning, and collects soak solution; Steep again with method and to wash 3 times, discard root system.Raffinate and earth are through centrifugal 2 min of 2000 rpm, and abandoning supernatant adds 494 g/L sucrose, and centrifugal 2 min of 2000 rpm clean supernatant liquor behind the mixing with 800 purpose separation sieves, collect larva and worm's ovum on the separation sieve.Washing lotion is used 100,200 and 500 repeatedly rinsings of purpose separation sieve again, collects gleanings on the 500 order separation sieves-major part and is worm's ovum; Filtered solution on the 500 order separation sieves cleans through 800 purpose separation sieves again, collects polypide-2 instar larvae on the separation sieve.The worm that separates and worm's ovum are stored in 4 ℃ the refrigerator standby.
3. experimental technique
Soaked with liquid method: specimen solution, the 200 μ l that get 10 times of 5 ml dilutions contain ± and the suspensions of 200 nematodes is in the culture dish of 6 cm in diameter, behind the mixing culture dish room temperature placed gently.Under anatomical lens, select at random 10 visuals field timings (6 hours, 12 hours, 24 hours) to observe, count disease borer population (motionless as standard with nematode) and bus borer population.Each specimen is established three repetitions, and take the substratum of inoculating strain not as contrast, obtains specimen to the lethality rate of nematode, just evaluates sample to the virulence of nematode with lethality rate.
Three lattice flat band methods: get 5 ml fermented liquids in three lattice plates, one lattice, all the other two lattice respectively add entry agar, add respectively nematode (about 100) at the water agar surface.Observe in microscopically timing (12 hours, 24 hours, 48 hours), the reactionless nematode of stiff acupuncture that observes is counted dead worm, writes down dead borer population and the bus borer population observed, calculates each fermented liquid to the lethality rate of nematode.Each processes triplicate, with equivalent LB substratum in contrast.
Active-fermented broth of the present invention is used in preparation poisoning Meloidogyne incognita (M. incognita) biological pesticide.
The present invention has good toxic action to Meloidogyne incognita (M. incognita), and Comprehensive Traits is good, and the advantage of high-efficiency low-toxicity can as the application of preparation biological pesticide nematode killing agent, be easy to suitability for industrialized production.
Embodiment:
The below is described in further detail the present invention with embodiment, but content of the present invention is not limited to this.
Embodiment one:
Bacillus proteus (Proteus sp.) CD-126 100 μ l after the activation are inoculated in (the bottled 250 ml substratum of 500 ml triangles) in the liquid LB substratum, under 28 ℃, 180 rpm conditions, cultivated 96 hours.Nutrient solution 8500 rpm after centrifugal 10 minutes, are got supernatant as specimen, dilute ten times afterwards with soaked with liquid method test activity.The results are shown in Table 1.
The activity that table 1 Bacillus proteus (Proteus sp.) CD-126 fermented liquid is measured Meloidogyne incognita (M. incognita) soaked with liquid method
The result shows: fermented liquid of the present invention has strong toxic action to Meloidogyne incognita (M. incognita), and the fermentation broth liquor infusion method measurement result after diluting 10 times shows that 24 hours lethality rates to Meloidogyne incognita (M. incognita) reach 93.53%.
Embodiment two:
Substantially with embodiment one, difference is the bottled 100ml substratum of 250 ml triangles when being fermentation, and 37 ℃, 120 rpm were cultivated 96 hours.
The activity that table 2 Bacillus proteus (Proteus sp.) CD-126 fermented liquid is measured Meloidogyne incognita (M. incognita) soaked with liquid method
The result shows: fermented liquid of the present invention has strong toxic action to Meloidogyne incognita (M. incognita), and the fermentation broth liquor infusion method measurement result after diluting 10 times shows that 24 hours lethality rates to Meloidogyne incognita (M. incognita) reach 99.29%.
The test of this example is identical with bacterial strain and substratum that precedent adopts, but volume of culture, temperature, rotating speed and asynchronism(-nization).Precedent is to be the bottled 250 ml substratum of 500 ml triangles, 28 ℃, 180rpm cultivation, and this example then is the bottled 100 ml substratum of 250 ml triangles, 37 ℃, 120rpm cultivation.Adopt the test of soaked with liquid method, test result shows that bacterial strain fermentation liquor is activity stabilized under different culture condition.
Embodiment three:
Substantially with embodiment two, difference is the bottled 50 ml substratum of 250ml triangle when being fermentation, and 30 ℃, 120 rpm are cultivated 96 hours fermented supernatant fluid as specimen, use the test of three lattice flat band methods.Activity the results are shown in Table 3.
The activity that table 3 Bacillus proteus (Proteus sp.) CD-126 fermented liquid is measured Meloidogyne incognita (M. incognita) three lattice flat band methods
The result shows: fermented liquid of the present invention has better toxic action to Meloidogyne incognita (M. incognita), and its fermenation raw liquid non-contact testing reached 98.32% to the lethality rate of Meloidogyne incognita (M. incognita) in the time of 24 hours.
The test of this example is identical with bacterial strain and substratum that precedent adopts, but volume of culture and temperature are different.Precedent is to be 37 ℃ of cultivations of the bottled 100 ml substratum of 250 ml triangles, and this example then is 30 ℃ of cultivations of the bottled 50 ml substratum of 250 ml triangles.Testing method has also adopted diverse ways, and precedent is the soaked with liquid method, and this example then is three lattice flat band methods, and fermented liquid does not directly contact with polypide, shows that the activeconstituents in the fermented liquid is volatile.
Claims (2)
1. actives is characterized in that this actives obtains through following steps:
A. Bacillus proteus (Proteus sp.) CD-126 that with preserving number is CGMCC No.6357 is kept in yeast extract Tryptones (LB) substratum, substratum is yeast extract: 5.0g, Tryptones: 10.0g, sodium-chlor: 10.0g, water 1000ml, and pH:7.0, in same liquid nutrient medium, activate 24 hours during use;
B. bacterial classification 100 μ l are inoculated in and are equipped with in the 50ml-250ml liquid LB substratum triangular flask after will activating, and are 28 ℃-37 ℃ in temperature, and rotating speed is to cultivate 96 hours under the condition of 120-180rpm, obtains nutrient solution; With nutrient solution 8500rpm after centrifugal 10 minutes, getting supernatant, to put into 4 ℃ refrigerator for subsequent use.
2. actives claimed in claim 1 is preparing the application of killing in Meloidogyne incognita (M.incognita) biotechnological formulation.
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| Application Number | Priority Date | Filing Date | Title |
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| CN 201210580357 CN103014068B (en) | 2012-12-28 | 2012-12-28 | Active matter and application thereof |
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| CN 201210580357 CN103014068B (en) | 2012-12-28 | 2012-12-28 | Active matter and application thereof |
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| CN103014068A CN103014068A (en) | 2013-04-03 |
| CN103014068B true CN103014068B (en) | 2013-10-30 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103484500B (en) * | 2013-08-29 | 2015-04-01 | 云南大学 | Bacterial CD-126 fermentation solution and application thereof |
| CN115057897B (en) * | 2022-06-24 | 2023-05-23 | 云南大学 | A preparation method for preparing two kinds of glucopyranosides by using nematode culture |
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| CN1311750C (en) * | 2006-02-14 | 2007-04-25 | 云南大学 | Active-fermented broth and use thereof |
| JP5168687B2 (en) * | 2007-12-25 | 2013-03-21 | 独立行政法人農業・食品産業技術総合研究機構 | How to protect against plant diseases caused by root-knot nematodes |
| CN101445785B (en) * | 2008-12-23 | 2011-04-13 | 北京市农林科学院 | Aspergillus niger fungus capable of poisoning plant parasitic nematodes, preparation method and application thereof |
| CN101787376B (en) * | 2010-01-05 | 2012-05-23 | 云南大学 | Paecilomyces varioti metabolite and application thereof |
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