CN1320105C - Bacillus for killing plant parasitic nematode and its preparation process and use thereof - Google Patents

Abstract

The present invention relates to a bacillus for poisoning plant nematodes and a preparation method and application thereof, which belongs to the technical field of microbial pesticide. A production strain of the present invention is bacillus nematocidus B16, and is prepared by a conventional method. The formula of a liquid culture medium is composed of the ingredients of 1 to 3% of yeast extracts, 0.5 to 2% of peptone, 1 to 4% o f glucose and 1 to 3% of NaCl. The pH value is from 6 to 7. The main active nematicide ingredients of the strain of the present invention are erzymes, and after the strain is fermented in liquid, the effective nematicide ingredients are extracted from metabolic products according to a conventional method for extracting the erzymes. The bacillus nematocidus B16 of the present invention has obvious poisoning activity to panagrellus redivivus nematodes and pine wood nematodes, and simultaneously, the bacillus grows fast under the culture conditions of the present invention, and is easily produced in an industrial way; the present invention has good prospects for application and development.

Description

A kind of food nematode genus bacillus and application thereof

Technical field:

The present invention relates to a kind of food nematode genus bacillus and application thereof, the microorganism belonging to genus technical field of pesticide.

Background technology:

Plant nematode is one of main Agricultural pests.Areal distribution is wide, host type is various because plant nematode has, and make plant secondary fungi or infectation of bacteria that disease and pest takes place easily, further increase the weight of plant hazard, therefore cause the whole world to surpass 1,000 hundred million dollars loss every year according to incomplete estimation plant nematode.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. takes place, and the oriented whole nation trend of stretching.Though can control the oxyuriasis of certain plants by approach such as traditional crop rotation, soil improvements, its application is subjected to the region easily and nematode itself has the influence of factors such as long-term latent; Our institute is main at present relies on chemical prevention and control method because most of chemical insecticides are for high poison, high residue or the agricultural chemicals that damages the ozone layer, along with the prolongation of duration of service, its drawback increasingly significant, restriction progressively the use of chemical insecticide.Therefore, the pathogenic nematode biological control comes into one's own day by day.Mainly be the fungi nematocides in the nematode biological prevention and control agent of researching and developing in the world at present, because the influence of the factors such as mycostasis that soil exists has limited the development of nematode biological and ecological methods to prevent plant disease, pests, and erosion.Simultaneously, in microorganism family, bacterium is the most sophisticated kind of suitability for industrialized production technology, and research and development bacterium nematocides is subjected to the various countries scientist and pays close attention to.

Summary of the invention:

The objective of the invention is to overcome the prior art deficiency, by a strain being had the genus bacillus research of intoxicating plant parasitical eelworm function, the biological nematocides of exploitation bacterial origin.

The bacterium that the present invention adopts is food nematode genus bacillus (Bacillus nematocidus) B16 bacterial strain, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on April 5th, 2004; Preservation is registered on the books and is numbered CGMCC No.1128.

Food nematode genus bacillus (Bacillus nematocidus) be the inventor carry out extracellular protease to the nematode infection Study on Mechanism in, from a large amount of soil bacterias, screen 1 novel species of bacillus.Discover the cytotoxicity that it is high to the nematode tool, after definite its activity of multiple batches of eelworm-killing activity test, entrust Wuhan University China typical culture collection center (CCTCC) that bacterial strain is identified, be defined as eating nematode genus bacillus novel species, Bacillus nematocidus.

The main morphological specificity of bacterial strain is: elongated rod shape, and individuality is bigger, and how the blunt circle in two ends exists to be dispersed in form.Bacterium colony is rounded in the dull and stereotyped training of LB, surface irregularity tarnish, similar fine powder shape, lawn white.The statospore of cultivation visible only a few more than 72 hours forms or is discharged into the gemma outside the born of the same parents on the LB substratum, and thalline almost generates gemma entirely after 5 days.Gemma is a long column shape, and inferior end is given birth to.

The present invention adopts is the comprehensive proterties of isolated 1 strain bacterial strain B16 preferably from food nematode bacillus nematocidus novel species; in view of this bacterial strain may be because (spontaneous, physics or chemical) sudden change, protoplastis fusion; conversion or other change by biotechnology or are transformed, and the mutant strain of any bacterial strain of this bacterial classification, mutation and derivative are all in protection scope of the present invention.

B16 bacterial strain poisoning nematode Study on mechanism is found that its nematode killing function mainly comes from albumen, enzyme composition.The main mechanism of action is that the extracellular protease that bacterial strain produces decomposes the nematode body wall, and failure line polypide wall complete rushes down the nematode content outward, and nematode kills the most at last.In whole nematicide process, be accompanied by and produce the effect of proteolytic enzyme, chitinase nematode, its virulence is remarkable.

The present invention is achieved in that

Enlarged culturing B16 bacterial strain extracts nematicide effective constituent, carries out the test of nematicide drug effect, determines its effect to nematode.

B16 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; 1-3%NaCl; 1.8-2% agar; PH6-7.The B16 thalline is inoculated on the substratum, cultivated 1-3 days down, obtain the test tube kind for 28-32 ℃;

2, fluid enlargement culture

The liquid culture based formulas is: the 1-3% yeast extract; The 0.5-2% peptone; 1-4% glucose; 1-3%NaCl; PH6-7, remainder is a water.The test tube kind is inoculated in the 500ml triangular flask liquid nutrient medium, and every bottled 200ml cultivated incubation time 2-5 days in 25-37 ℃ of following shaking table, and rotating speed is 200-300rpm.

3, extract the eelworm-killing activity composition

With fermenting culture centrifugal 10 minutes in 5000rpm/min, get supernatant liquor, press the 0%-100% saturation ratio and add ammonium sulfate, 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, 000-14000Da) 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 B16 eelworm-killing activity:

1, preparation test with medicament

Cultivate the B16 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 B16 bacterial strain in the substratum.

Contrast 2: in contrast with the 50mM phosphoric acid buffer.

Contrast 3: in contrast with the dialysis solution after the dialysis

Contrast 4: in order to prove the nematicide effective ingredient is albumen, will prepare reagent agent and boil after the deactivation in contrast.

3, 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.

4, test-results

Table 1 B16 is to Panagrellus redivivus nematode toxic 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
							Test with medicament contrast 1 contrast 2 contrasts 3 contrasts 4	60 10 10 10 20	The stiff no change no change of polypide no change no change	90 20 10 15 20	Not not not dissolving of dissolving of dissolving of dissolving of dissolving appears in body wall	98 20 20 20 30	The complete body wall complete body of most dissolving body wall complete body wall wall is complete

Table 2 B16 is to pine wood nematode Bursaphelenchus xylophilus toxic 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
							Test with medicament contrast 1 contrast 2 contrasts 3 contrasts 4	70 5 5 5 20	The stiff no change no change of polypide no change no change	90 20 10 10 20	Body wall is coarse, begins to occur not not not dissolving of dissolving of dissolving of dissolving of dissolving	95 30 15 20 30	Partly not not not dissolving of dissolving of dissolving of dissolving of dissolving

The result shows, the test with medicament of extracting from B16 strain liquid tunning is to Panagrellusredivivus, the equal tool of Bursaphelenchus xylophilus nematode insecticidal effect preferably, and the average mortality of 48 hours nematodes is more than 90%.To handle the back nematode and observe under light microscopic, compared with the control, experimental group nematode body wall is dissolved fully by complete part dissolving one and changes obviously.Because the destruction of nematode body wall, a large amount of outer the rushing down of nematode content cause nematode finally dead.But because for examination nematode difference, its action time is also inequality.36 hours mortality ratio of Panagrellus redivivus nematode have reached 98%, and pine wood nematode death in 60 hours reaches 95%.

The result shows that simultaneously in the B16 bacterial strain, nematicide main active ingredient is an enzyme.And will be with batch test with medicament of extracting (contrast 4) 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 also further proved conclusively B16 the active ingredient of nematode has been mainly enzyme.

B16 bacterial strain of the present invention is the brand-new food nematode genus bacillus of finding with significant eelworm-killing activity of a class, and it mainly acts on composition is enzyme.The B16 bacterial strain is grown under culture condition of the present invention soon simultaneously, carries out suitability for industrialized production easily, has possessed good application and development prospect.

Embodiment:

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

Embodiment one:

To eat nematode genus bacillus (Bacillus nematocidus) B16 thalline and be inoculated on the test tube nutrient agar inclined-plane, culture medium prescription is: 2% yeast extract, 1.5% peptone; 2% glucose; 1-3%NaCl; 1.8% agar; PH7.The B16 thalline 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 extract; 1.5% peptone; 2%NaCl; 3% glucose; PH7, remainder is a water.Cultivated incubation time 3 days in 30 ℃ of shaking tables, rotating speed is 220rpm.

With fermenting culture centrifugal 20 minutes in 8000rpm/min, get supernatant liquor, add ammonium sulfate in the ratio of 100% saturation 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, 000-14000Da) 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 extract; 0.5% peptone; 1%NaCl; 1% glucose.

Embodiment three:

Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is: 1% yeast extract; 2% peptone; 3%NaCl; 4% glucose.

Claims (2)

1, a kind of food nematode genus bacillus, it is food nematode genus bacillus (Bacillus nematocidus) CGMCCNo.1128.

2, the application of the described food of claim 1 nematode genus bacillus is characterized in that this food nematode genus bacillus is used for the biological control to plant Panagrellus redivivus nematode and pine wood nematode.