CN1663394A - Wireworm-killing biologic bacterial agent, its preparation and application - Google Patents

Abstract

The present invention relates to a process for preparing and application of bionematocide and belongs to technique domain of biological pesticide. Product of the invention can be gained from conventional method that the produced strain is cultivated in test tube, enlarged cultivated in liquid and extracts active constituents of nematocide, whose produced strain is Lecanicillium psalliotae YMF1.00112, with conservation number CGMCC No.1312. The formulation of enlarged liquid base in the invention is, dextrose 10g, ammonia sulfate 1g, magnesium sulfate 1g, ferrous sulfate 0.015g, leachate of potato 1000mg, pH6.5. the invention can poison pine wood nematode and Panagrellus redivivus and be used as bio-prevention to plant parasitic nematode. The product has the advantage of high poison effect, safety to human body and environment protection, etc.

Description

A kind of wireworm-killing biologic bacterial agent and its production and application

Technical field:

The present invention relates to a kind of wireworm-killing biologic bacterial agent and its production and application, belong to biological pesticide technical field.

Background technology:

Plant nematode is the plant disease that generally takes place in a kind of world wide, and only the known kind of root-knot nematode reaches kind more than 70, endangers 3000 various plants, and cause damage huge every year.According to FAO (Food and Agriculture Organization of the United Nation) statistics, the loss that cause because of nematode in the annual whole world is up to more than 1,000 hundred million dollars, and the loss that calendar year 2001 China crop nematode harm causes is up to 23,300,000,000 yuan.Pine wood nematode is called as smokeless forest fire, in Jiangsu, partly the area is serious Zhejiang, Guangdong, Shandong, Anhui, Hubei, Shanghai, Taiwan, Hong Kong etc. that the trend of stretching in the oriented whole nation takes place.More seriously, cause the crop root disease seriously to take place because root-knot nematode is caused the crop wound after infecting.

At present to nematoda control still based on chemical nematocide.For a long time, chemical nematocide is prevented and treated and has been brought into play important function in the nematodosis ensureing agricultural production.But along with progress of science and technology, find that many chemical nematocides all have side effect, not only develop immunity to drugs, more cause environmental pollution, be detrimental to health, have many disabled or be about to forbidding in succession.Celfume, methylene bromide have been banned use of as the U.S..The chemistry nematocide when ensureing agricultural production, has brought certain negative effect for human health and environment at the control eelworm harm, and exploitation high-efficiency low-toxicity nematocide and biological nematocide are extremely urgent.

Verticillium dahliae is the very important biocontrol fungi of a class, wherein much all is the disease fungus of important insect and nematode, can multiple harmful insect of effectively preventing and nematode, in the biological control of the cause of disease insect of plant and nematode, play important effect.Wherein, Verticillium lecanii and thick spore Pu Keniya bacterium have been applied to the exploitation of biological insecticides, and the effect of insect and nematode are had not yet to see report about cutter spore Verticillium dahliae.

Summary of the invention:

The objective of the invention is by a strain being produced the research that extracellular enzyme also can effectively decompose the inner parasitic epiphyte cutter spore Verticillium dahliae of nematode body wall structure, exploitation wireworm-killing biologic bacterial agent.

The inventor screens a strain plant nematode is had the inner parasitic epiphyte-cutter spore Verticillium dahliae Lecanicillium psalliotae of fine toxic effect, YMF1.00112.The YMF1.00112 bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on January 27th, 2005; The numbering CGMCC No.1312 that preservation is registered on the books.

Cutter spore Verticillium dahliae Lecanicillium psalliotae, YMF1.00112 are the strain inner parasitic epiphyte that the inventor separates from the nematode body surface, and main morphological feature is:

Growth is very fast on the PDA medium, cultivates that colony diameter reaches 3～6cm after 7 days, the mycelial growth densification, and colony colour is white in color, and the back side is pale yellow extremely brown; The conidiophore colyliform and is given birth to, and wide 13～29 μ m of base portion, wide 0.2～0.5 μ m in top, conidium head and give birth to, ellipse, and sickleshaped, 0.6～2.2 * 2.0～9.0 μ m do not see chlamydospore.

After the present invention adopts conventional method with cutter spore Verticillium dahliae enlarged culture, extract nematicide active ingredient, measure its insecticidal effect saprophitic nematode (Panagrellus redivivus) and pine wood nematode (Bursaphelenchus xylophilus).

The present invention is achieved in that

YMF1.00112 strain culturing method:

1. the test tube kind is cultivated

The test tube kind adopts conventional method to cultivate, and the YMF1.00112 mycelium is inoculated on the PDA medium, cultivates 7～10 days down, obtains the test tube kind for 20-25 ℃.

2. fluid enlargement culture

The liquid culture based formulas is: 8-15g glucose; 0.8-1.5g ammonium sulfate; 0.4-0.6g magnesium sulfate; 0.01-0.02g ferrous sulfate; 1000ml potato diffusion juice (the 100g potato adds the 1200ml water boil and filters, and water complements to 1000ml), pH6.5.Use the 250ml triangular flask, each dress 60ml culture fluid, 121 ℃ of sterilizations insert the test tube kind after 20 minutes, and 26 ℃, shaking table was cultivated 6 days under the 200rpm.

Extract the eelworm-killing activity composition:

After bacterium liquid that the aforementioned liquids enlarged culture is good filters, concentrate with the milipore filter in 5KDa aperture.The concentrate dialysis treatment is carried out purifying with cation exchange column later, collect respectively and penetrate peak and eluting peak, biological activity determination shows that most of proteinase activity is at eluting peak, merge eluting peak again with the further purifying of drainage column, collect eluting peak and handle with distill water dialysis, it is pure that electrophoretic analysis has reached electrophoresis.Freeze drying is the sample freeze-drying and be dissolved in the distilled water of 2ml, and-20 ℃ of preservations concentrate later preparation through milipore filter and are mematocide.

The eelworm-killing activity test:

1. with medicament and contrast with medicament are tested in preparation

1) preparation test with medicament

Obtain bacterium liquid by aforementioned YMF1.00112 strain culturing method, by the method preparation test with medicament of aforementioned extraction eelworm-killing activity composition.

2) preparation contrast with medicament

Contrast 1: by the preparation of preparation test administrated method, different is not insert the YMF1.00112 bacterial strain in the culture fluid;

Contrast 2: will prepare 100 ℃ of reagent agents and boil after 30 minutes in contrast, to prove that the nematicide effective ingredient is a protease.

2. nematode is used in the preparation test

1) preparation Panagrellus redivivus nematode

The P.redivivus nematode is inoculated on the medium oatmeal, cultivated 6 days down, freeze in 4 ℃ of refrigerators standby in 28 ℃.Required nematode is washed out with the graceful funnel method of shellfish, place in the 5ml centrifuge tube, add the washing of 5ml sterile water, instantaneous centrifugal, abandon supernatant, repeat 5 times and obtain clean for the examination nematode.Is that content is the nematode suspension of 15/μ l with sterile water with the nematode dilution.

2) preparation pine wood nematode (Bursaphelenchus xylophilus)

Put into 15g through 2 days iblet of water logging bubble in the 100ml triangular flask, add water 10ml, autoclaving inserts Botrytis cinerea (Botrytis cinerea).Cultivated 4 to 7 days for 25 ℃.After treating that mycelia is paved with triangular flask, inoculation was cultivated 15 to 20 days for 28 ℃ through the pine wood nematode of 0.25% clorox surface sterilization.With sterile water nematode is washed, making content is the nematode suspension of 15/μ l.

3. test method

1) test of pesticide effectiveness method

Get test with medicament 200 μ l in the 1.5ml centrifuge tube, add 60 of living nematodes, centrifuge tube keeps flat, and places under 25 ℃, and Panagrellus redivivus nematode was respectively at 12 hours, 20 hours; Pine wood nematode is respectively at the lethality of checking and calculated nematode in 12 hours, 20 hours.Identify that dead method is: add 1-5 and drip 5%Nacl solution in handling centrifuge tube, observe after 2 minutes, dead worm is stiff, and the worm that lives is then curled or twisting.

Carry out control experiment with 2 kinds of contrast medicaments respectively.

The test establish three parallel, twice repetition.

2) nematocidal effect of purification of samples and to the decomposition of nematode body wall: experimental technique is the same, with the sample of the observation by light microscope purifying decomposition to the nematode body wall.

4. result of the test

12 couples of Panagrellus redivivus of table 1:YMF1.001 nematode insecticidal effect

Table 2:YMF1.00112 is to the insecticidal effect of pine wood nematode (Bursaphelenchusxylophilus)

The result shows, the test with medicament of extracting from YMF1.00112 strain liquid tunning is to Panagrellusredivivus nematode and the equal tool of pine wood nematode nematicide effect preferably.Handle nematode with 5 times of concentrated samples as soup, 100% death of Panagrellus redivivus nematode and nematode are decomposed fully after 12 hours, the pine wood nematode lethality also reaches more than 60% (table 2), the lethality of pine wood nematode also reaches 100% after 20 hours, but the body wall structure of pine wood nematode is complete, is not decomposed.Experimental result shows simultaneously, is lower than thick concentrating sample through the eelworm-killing activity of the sample of cation seperation column purifying, illustrated that other composition has participated in nematicide process.

From handling the variation of nematode, experienced from the part body wall decomposing all processes of degraded of polypide, illustrate that the active ingredient master is for will decompose the enzyme of nematode.And will be with batch test with medicament of extracting through boiling processing, cause protein denaturation after, its eelworm-killing activity is very little, and dissolution phenomena do not appear in the nematode body wall, this has proved that also the YMF1.00112 bacterial strain is mainly enzyme to the active ingredient of nematode.

Cutter spore Verticillium dahliae YMF1.00112 of the present invention is a kind of fungi that nematode is had better toxic action function, it mainly acts on composition is extracellular enzyme, to nematode particularly the virulence of pine wood nematode be significantly higher than or, possessed good application and development prospect with the thick spore Pu Keniya bacterium of report before equaling.

Embodiment:

Below be embodiments of the invention, but content of the present invention is not limited thereto.

Embodiment one:

The YMF1.00112 inoculation to the PDA inclined-plane, was cultivated 8 days down, obtained the test tube kind for 20-25 ℃.

The test tube kind is inoculated in the 250ml triangular flask liquid nutrient medium, every bottled 60ml, the liquid culture based formulas is: 10g glucose, 1g ammonium sulfate, 0.5g magnesium sulfate, 0.015g ferrous sulfate, 1000ml potato diffusion juice, pH6.5.Each 250ml triangular flask packing 60ml culture fluid, 121 ℃ of sterilizations insert the test tube kind after 20 minutes, and 26 ℃, shaking table was cultivated after 6 days under the 200rpm, and bacterium liquid is filtered, and concentrated with the milipore filter in 5KDa aperture.The concentrate dialysis treatment is carried out purifying with cation exchange column later, collect respectively and penetrate peak and eluting peak, biological activity determination shows that most of proteinase activity is at eluting peak, merge eluting peak again with the further purifying of drainage column, collect eluting peak and handle with distill water dialysis, it is pure that electrophoretic analysis has reached electrophoresis.Freeze drying is the sample freeze-drying and be dissolved in the distilled water of 2ml-20 ℃ of preservations.Concentrate later preparation through milipore filter and be mematocide.

Embodiment two:

Substantially with embodiment one, difference is: the liquid culture based formulas is a 8g glucose; 1.5g ammonium sulfate; 0.6g magnesium sulfate; 0.01g ferrous sulfate.

Embodiment three:

Substantially with embodiment one, difference is: the liquid culture based formulas is: 15g glucose; 0.8g ammonium sulfate; 0.4g magnesium sulfate; 0.02g ferrous sulfate.

Result of use of the present invention sees table 1 and table 2 for details.

Claims (3)

1, a kind of wireworm-killing biologic bacterial agent, this microbial inoculum obtains through the cultivation of test tube kind, fluid enlargement culture and after extracting eelworm-killing activity composition operation by producing bacterial strain and auxiliary material, the production bacterial strain that it is characterized in that this bacteria agent is: cutter spore Verticillium dahliae (Lecanicillium psalliotae) YMF1.00112, and the YMF1.00112 bacterial strain is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation date: on January 27th, 2005; Preserving number CGMCC No.1312.

2, the preparation method of the described bacteria agent of claim 1 comprises the cultivation of test tube kind, fluid enlargement culture and extracts eelworm-killing activity becoming operation break-down, it is characterized in that the prescription of fluid enlargement culture base is: 8-15g glucose; 0.8-1.5g ammonium sulfate; 0.4-0.6g magnesium sulfate; 0.01-0.02g ferrous sulfate; 1000ml potato diffusion juice, pH6.5.

3, the said biologic product of claim 1 is characterized in that said preparation has toxic action to Panagrellus redivivus nematode and pine wood nematode, can be used for the plant nematode biological and ecological methods to prevent plant disease, pests, and erosion.