CN1194085C - Bacillus pumilus for killing nematoda and its preparation and use - Google Patents

Abstract

The present invention relates to side spore bacillus brevis with nematicide function and a preparation method and the application of the side spore bacillus brevis with the nematicide function, which belongs to the technical field of microbial pesticide. A production strain of the present invention is erevibacillus laterosporus G4 and is prepared by a conventional method. The formulation of a liquid fermentation medium comprises the ingredients of 1 to 3% of yeast extract powder, 10 to 20% of soybean meal powder, 0.5 to 2% of fish meal, 1 to 4% of glucose and water as the rest; the pH value is 7. The method for extracting the effective nematicide ingredients from a liquid fermentation product comprises the following steps that a fermented culture is centrifugated for 20 mintues at the speed of 8000 rpm/min, supernatant liquid is taken, ammonium sulfate is added according to the proportion of 1 to 0.85, and the supernatant liquid is statically placed for two hours at the temperature of 4 DEG C and is centrifugated for 30 mintues at the speed of 8000 rpm/min; the supernatant liquid is abandoned; the ammonium sulfate is solved anew in 50mM of phosphate buffer liquid, a solution obtained by resolution is filled in 21mm of dialysis bag and is dialyzed for 3 to 4 times in 50mM of the phosphate buffer liquid of 10 to 20 times, and the dialysis takes 3 hours each time. In the present invention, the effective nematicide ingredients are extracted from metabolic products after the strain is fermented in the liquid, and a biologic nematicide is prepared. The product of the present invention has the advantages of high toxicity to pine wood nematodes and good application prospects.

Description

A kind of microbial inoculum and preparation method and application with nematode killing function

Technical field:

The present invention relates to a kind of microbial inoculum and preparation method and application, the microorganism belonging to genus technical field of pesticide with nematode killing function.

Background technology:

Plant nematode is the Plant diseases that generally takes place in a kind of world wide, and only the known kind of root knot nematode reaches kind more than 70,3000 various plants that cause harm, and the loss that causes every year is huge.According to Food and Argriculture OrganizationFAO's statistics, cause damage the whole world up to 1,000 hundred million dollar because of nematode every year.Main cash crop such as China nematode main harm tobacco, flowers, vegetables, cotton, soybean, peanut, pseudo-ginseng, Radix Panacis Quinquefolii become the critical limitation factor that develops these crops.According to the incomplete statistics of national tobacco disease pest control information center, calendar year 2001 whole nation tobacco root knot nematode onset area reached 550,000 hectares, more than 500,000,000 yuan of direct economic loss.Only Yunnan Province's onset area just reaches 2.7 ten thousand public affairs and inclines, 1.2 hundred million yuan of direct economic losses, and indirect loss is even more serious.The Heilongjiang Province only the soybean Cyst nematode cause every year with a toll of 800,000,000 yuan more than.Pine wood nematode is called as smokeless hill 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.In Shandong, vegetables main producing region such as Guangdong, Hainan and Yunnan, root knot nematode is in rising trend.As the cucumber in Shandong cause because of using in a large number that the eater suffers from diarrhoea behind the chemical nematocides, root knot nematode takes place and have to stop production because of serious in Guizhou Province's Radix Panacis Quinquefolii.More seriously, cause the crop root disease seriously to take place owing to after root knot nematode infects, cause the crop wound.The pseudo-ginseng root-rot in Yunnan is exactly an exemplary.The chemistry nematocides has been brought into play vital role in control of nematode since the eighties of last century discovery forties.Along with progress of science and technology, find that many chemical nematocidess all have side effect, cause environmental pollution, be detrimental to health.Have many disabled or be about to forbidding in succession.Particularly plant nematode betides crop root, because the singularity of edatope, must heavy dose uses and could guarantee its preventive effect.And heavy dose of chemical pesticide is very serious to groundwater pollution.Therefore, research and development high-efficiency low-toxicity low residue nematocides has been the important topic in the agricultural sustainable development.

Side spore bacillus brevis is that a class is aerobic, can form the bacterium of gemma.Early-stage Study shows that side spore bacillus brevis can form parasporal crystal and have insecticidal function in its life history.And the major ingredient of parasporal crystal is an insecticidal crystal protein, and insecticidal crystal protein has good poisoning function to insect.But in side spore bacillus brevis, do not produce parasporal crystal, produce insecticidal function by enzyme, toxalbumin or other phallotoxins and have not yet to see report.

Summary of the invention:

The objective of the invention is to produce the side spore bacillus brevis research that enzyme decomposes the nematode function, develop biological nematocides by a strain is had.

The strain that the present invention screens has the side spore bacillus brevis Brevibacillus laterosporus G4 of fine insecticidal function to plant nematode, and the G4 bacterial strain has been deposited in Chinese typical culture collection center; Address: China. Wuhan. Wuhan University; Preservation date: on June 2nd, 2003; The numbering CCTCC NO:M203045 that preservation is registered on the books.

Brevibacillus laterosporus G4 strain morphology of the present invention is characterized as: elongated rod shape, and individuality is bigger, and how the blunt circle in two ends exists with the aggegation form.Rounded, the protuberance of bacterium colony, edge are more neat in the dull and stereotyped training of YPD, mattness, coarse, similar fine powder shape, lawn white.The statospore of cultivating visible only a few more than 36 hours on the YPD substratum forms or is discharged into the gemma born of the same parents outside, and at NA or LB culture medium culturing 12-16 hour, the just visible gemma that comes off in a large number, gemma is a paddle shape body in the ellipse.The difference that G4 bacterial strain and other have the side spore bacillus brevis maximum of insecticidal function is not form parasporal crystal, and its nematode killing function that has mainly is made of compositions such as albumen, enzymes.Its nematicide mechanism reason is also different fully.G4 at first forms crude protein and decomposes the nematode body wall, and in body wall infection thread polypide, nematode is all decomposed the most at last.In whole nematicide process, be accompanied by and produce the effect of proteolytic enzyme, chitinase nematode, its virulence is significantly higher than similar side spore bacillus brevis.

The inventor isolates a strain side spore bacillus brevis G4 from the genetic engineering bacterium that makes up in carrying out the research of nematicide alkalescence extracellular protease, find the insecticidal activity that it is high to the nematode tool.After carrying out multiple batches of eelworm-killing activity test behind the bacterial strain purifying, entrust Wuhan University China typical culture collection center (CCTCC) that bacterial strain is identified, be defined as side spore bacillus brevis Brevibacillus laterosporus G4.

The present invention is achieved in that

Enlarged culturing G4 bacterial strain extracts nematicide effective constituent, carries out the test of nematicide drug effect, determines its effect to nematode.

G4 cultural method (below be weight percentage):

1, the test tube kind is cultivated

Culture medium prescription is: 1-3% yeast extract, 0.5-2% peptone; 1-4% glucose; 1.8-2% agar; PH6-7.The G4 mycelium is inoculated on the substratum, cultivated 1-4 days down, obtain the test tube kind for 28-32 ℃;

2, fluid enlargement culture

The liquid culture based formulas is: the 1-3% yeast powder; The 10-20% soybean-cake flour; The 0.5-2% fish meal; 1-4 glucose; PH6-7, remainder is a water.The test tube kind is inoculated in the 250ml triangular flask liquid nutrient medium, and every bottled 100ml cultivated incubation time 2-5 days in 25-35 ℃ of following shaking table, and rotating speed is 200-300rpm.

3, extract the eelworm-killing activity composition

With fermenting culture centrifugal 20 minutes in 8000rpm/min, get supernatant liquor, add ammonium sulfate in 1: 0.85 ratio, left standstill 2 hours in 4 ℃, 8000rpm/min is centrifugal 30 minutes then, abandons supernatant.(the 50mM SODIUM PHOSPHATE, MONOBASIC of 39ml adds the 50mM Sodium phosphate dibasic of 61ml with the 50mM phosphoric acid buffer, pH is 7) dissolving again, to dissolve the solution that the obtains 21mm dialysis tubing (MW:8 that packs into again, 000-14400KDa) in 10-20 50mM phosphoric acid buffer doubly, dialyse 3-4 time, each 3 hours, promptly get the nematicide soup.Be stored in 4 ℃ of refrigerators soup stand-by.

The test of G4 eelworm-killing activity:

1, preparation test with medicament

Cultivate the G4 bacterial strain by aforementioned liquids enlarged culturing method, by the method preparation test with medicament of aforementioned extraction eelworm-killing activity composition.

2, preparation contrast with medicament

Contrast 1: aforementioned 1 preparation test with medicament method preparation contrast with medicament, but do not insert the G4 bacterial strain in the substratum.

Contrast 2: in contrast with clear water.

Contrast 3: in order to prove the nematicide effective ingredient is albumen, will prepare reagent agent and boil after the deactivation in contrast.

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 sterilized 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 sterilized water with the nematode dilution.

(2) preparation pine wood nematode (Bursaphelenchus xylophilus)

Put into 15g through 2 days corn grain 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 sterilized 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 150 of living nematodes, centrifuge tube keeps flat, and places under 25 ℃, and Panagrellus redivivus nematode was respectively at 12 hours, 24 hours, 48 hours; Pine wood nematode is respectively at the mortality ratio of checking the calculating nematode in 24 hours, 48 hours, 60 hours.And under opticmicroscope, observe observation line polypide wall changing conditions

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.

Respectively with 3 kinds of contrast medicaments, every processing triplicate.

(2) G4 decomposes the nematode viewing test

Living nematode was handled respectively under 25 ℃ 12 hours and 24 hours with the test with medicament, sample after handling is washed three times with the PBS damping fluid, fixing with 3% glutaraldehyde with hungry acid, displace moisture with gradient glycerine, ethyl acetate is replaced once more, vacuum lyophilization, gold-plated film, be fixed at last on the slide glass, with scanning electron microscopic observation (SEM).Observe with the processing same period of contrast medicament.

4, test-results

Table 1 G4 is to Panagrellus redivivus nematode insecticidal effect

Chemicals treatment	The nematode death condition that is fixed
							12 hours		24 hours		36 hours
	Mortality ratio	Nematode changes	Mortality ratio	Nematode changes	Mortality ratio	Nematode changes
							The test with medicament	?85	Polypide is stiff	95	Dissolving appears in body wall	98	Most dissolving
Contrast 1	?10	No change	20	Not dissolving	30	Body wall is complete
							Contrast 2	?10	No change	10	Not dissolving	20	Body wall is complete
Contrast 3	?10	No change	10	Not dissolving	30	Body wall is complete

Table 2 G4 is to the pine wood nematode insecticidal effect

Chemicals treatment	The nematode death condition that is fixed
							24 hours		48 hours		60 hours
	Mortality ratio	Nematode changes	Mortality ratio	Nematode changes	Mortality ratio	Nematode changes
							The test with medicament	80	Polypide is stiff	95	Body wall is coarse, begins to occur dissolving	96	Partly dissolving
Contrast 1	?15	No change	20	Not dissolving	30	Not dissolving
							Contrast 2	?5	No change	10	Not dissolving	15	Not dissolving
Contrast 3	?20	No change	20	Not dissolving	30	Not dissolving

The result shows, the test with medicament of extracting from G4 strain liquid tunning is to Panagrellus redivivus nematode and the equal tool of pine wood nematode insecticidal effect preferably, and the average mortality of nematode is more than 95%.From handling the variation of nematode, experienced from the part body wall and dissolved (Fig. 3) to the whole dissolved processes of polypide (Fig. 1), 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, 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 G4 is mainly enzyme to the active ingredient of nematode.

G4 bacterial strain of the present invention is that a class obviously is different from the side spore bacillus brevis that other produce parasporal crystals, and it mainly acts on composition is enzyme, to nematode particularly the virulence of pine wood nematode be significantly higher than other same quasi-microorganisms of finding at present.The G4 bacterial strain is grown under culture condition of the present invention and is exceedingly fast simultaneously, has possessed good application and development prospect.

Description of drawings:

Fig. 1 display process nematode is decomposed situation.

Fig. 2 shows the contrast nematode that polypide remains intact.

Fig. 3 display process nematode head part is separated situation.

Fig. 4 shows the contrast nematode that head remains intact.

Embodiment:

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

Embodiment one:

Brevibacillus laterosporus G4 thalline is inoculated on the test tube nutrient agar inclined-plane, and culture medium prescription is: 2% yeast extract, 1.5% peptone; 2% glucose; 1.8% agar; PH7.The G4 mycelium is inoculated on the substratum, cultivated 2 days down, obtain the test tube kind for 30 ℃.

The test tube kind is inoculated into (every bottled 100ml) in the 250ml triangular flask liquid nutrient medium, and the liquid culture based formulas is: 2% yeast powder; 15% soybean-cake flour; 1% fish meal; 3% glucose; PH7, remainder is a water.Cultivated incubation time 3 days in 30 ℃ of following shaking tables, rotating speed is 220rpm.

With fermenting culture centrifugal 20 minutes in 8000rpm/min, get supernatant liquor, add ammonium sulfate in 1: 0.85 ratio, left standstill 2 hours in 4 ℃, 8000rpm/min is centrifugal 30 minutes then, abandons supernatant.(the 50mM SODIUM PHOSPHATE, MONOBASIC of 39ml adds the 50mM Sodium phosphate dibasic of 61ml with the 50mM phosphoric acid buffer, pH is 7) dissolving again, to dissolve the solution that the obtains 21mm dialysis tubing (MW:8 that packs into again, 000-14400KDa) in 10-20 50mM phosphoric acid buffer doubly, dialyse 3-4 time, each 3 hours, promptly get the nematicide soup.

Embodiment two:

Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is: 3% yeast powder; 10% soybean-cake flour; 2% fish meal; 1% glucose.

Embodiment three:

Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is: 1% yeast powder; 20% soybean-cake flour; 0.5% fish meal; 4% glucose.

Claims (3)

1, a kind of microbial inoculum with nematode killing function, 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, and it is characterized in that producing bacterial strain is side spore bacillus brevis (Brevibacillus laterosporus) G4, CCTCC NO:M203045.

2, the preparation method of the described microbial inoculum of claim 1 comprises the cultivation of test tube kind, fluid enlargement culture and extracts eelworm-killing activity composition operation, it is characterized in that:

2.1 liquid fermentation medium prescription: 1-3% yeast powder; The 10-20% soybean-cake flour; The 0.5-2% fish meal; 1-4 glucose; PH7, remainder is a water;

2.2 from liquid fermentation production, extract the method for nematicide effective constituent be: with fermenting culture centrifugal 20 minutes in 8000rpm/min, get supernatant liquor, add ammonium sulfate in 1: 0.85 ratio, left standstill 2 hours in 4 ℃, 8000rpm/min is centrifugal 30 minutes then, abandons supernatant, dissolves again with the 50mM phosphoric acid buffer, again the dissolving solution that the obtains 21mm dialysis tubing of packing into is dialysed each 3 hours 3-4 time in 10-20 50mM phosphoric acid buffer doubly.

3, the described microbial inoculum of claim 1 is characterized in that this microbial inoculum has toxic action to Panagrellus redivivus nematode and pine wood nematode, can be applied to the plant nematode biological and ecological methods to prevent plant disease, pests, and erosion.

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