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 bacterial agent with nematicide function, its preparation method and application

Technical field:

The invention relates to a fungus agent with a nematicide function, a preparation method and an application, and belongs to the technical field of microbial pesticides.

Background technique:

Plant parasitic nematodes are a common plant disease worldwide. There are more than 70 known types of root-knot nematodes alone, which damage more than 3,000 plants and cause huge losses every year. According to statistics from the Food and Agriculture Organization of the United Nations, nematodes cause global losses of up to 100 billion U.S. dollars every year. Nematodes in my country mainly harm tobacco, flowers, vegetables, cotton, soybeans, peanuts, Panax notoginseng, American ginseng and other major economic crops, and become an important limiting factor for the development of these crops. According to incomplete statistics from the National Tobacco Pest Prevention and Control Information Center, in 2001 the area of tobacco root-knot nematode incidence in the country had reached 550,000 hectares, and the direct economic loss was more than 500 million yuan. The affected area in Yunnan Province alone reached 27,000 hectares, and the direct economic loss was 120 million yuan, and the indirect loss was even more serious. In Heilongjiang Province, the annual loss caused by soybean cyst nematode alone is as much as 800 million yuan. Pine xylophilus is known as smokeless forest fires, which have seriously occurred in some areas such as Jiangsu, Zhejiang, Guangdong, Shandong, Anhui, Hubei, Shanghai, Taiwan, Hong Kong, etc., and have a tendency to spread to the whole country. In the main vegetable producing areas such as Shandong, Guangdong, Hainan and Yunnan, vegetable root-knot nematodes are on the rise. For example, the consumption of cucumbers in Shandong caused diarrhea due to extensive application of chemical nematicides, and the production of American ginseng in Guizhou Province had to be discontinued due to severe occurrence of root-knot nematodes. What's more serious is that after root-knot nematode infestation, crop wounds are caused, resulting in serious root diseases of crops. The notoginseng root rot in Yunnan is a typical example. Chemical nematicides have played an important role in nematode control since their discovery in the 1940s. With the advancement of science and technology, many chemical nematicides have been found to have side effects, causing environmental pollution and endangering human health. Many have been disabled or will be disabled one after another. In particular, plant parasitic nematodes occur in the roots of crops. Due to the particularity of the soil environment, it must be applied in large doses to ensure its control effect. However, large doses of chemical pesticides pollute groundwater very seriously. Therefore, the research and development of high-efficiency, low-toxicity and low-residue nematicides has become an important topic in the sustainable development of agriculture.

Brevibacillus sporogenes are a class of aerobic, spore-forming bacteria. Previous studies have shown that Brevibacillus spp. can form parasporal crystals in its life cycle and have insecticidal function. The main component of parasporal crystals is insecticidal crystal protein, which has a good poisoning function to pests. But in Brevibacillus lateralspora, parasporal crystals are not produced, and the insecticidal function produced by enzymes, toxic proteins or other toxic peptides has not been reported so far.

Invention content:

The purpose of the present invention is to develop a biological nematocide through the research on a strain of Brevibacillus sporogenes that has the function of producing enzymes to decompose nematodes.

Brevibacillus laterosporus G4, a strain of Brevibacillus laterosporus G4 screened by the present invention that has a good insecticidal function against plant parasitic nematodes, has been preserved in the China Type Culture Collection Center; address: China.Wuhan.Wuhan University; date of preservation : June 2, 2003; the number CCTCC No. of deposit registration: M203045.

The morphological characteristics of the Brevibacillus laterosporus G4 strain of the present invention are: long rod shape, larger individuals, blunt rounded ends, mostly in the form of agglutination. On the YPD plate culture, the colonies are round, raised, and the edges are relatively neat, the surface is matte, rough, similar to fine powder, and the bacterial lawn is white. When cultured on YPD medium for more than 36 hours, a very small number of endospores formed or released to the outside of the cell can be seen. After 12-16 hours of culture on NA or LB medium, a large number of shed spores can be seen, and the spores are oval and single. Canoe. The biggest difference between the G4 strain and other Brevibacillus lateralsporosa with insecticidal function is that it does not form parasporal crystals, and its nematicidal function is mainly composed of proteins, enzymes and other components. Its nematicide mechanism mechanism is also completely different. G4 first forms crude protein to decompose the nematode body wall, invades the nematode body through the body wall, and finally decomposes all nematodes. During the whole process of killing nematodes, along with the production of protease and chitinase, its virulence is significantly higher than that of the same species of Brevibacillus spp.

The present inventors isolated a strain of Brevibacillus sporogenes G4 from the constructed genetically engineered bacteria in the research of nematicidal alkaline extracellular protease, and found that it has extremely high insecticidal activity against nematodes. After the strain was purified and conducted multiple batches of nematicide activity tests, the China Center for Type Culture Collection (CCTCC) of Wuhan University was entrusted to identify the strain and it was determined to be Brevibacillus laterosporus G4.

The present invention is achieved like this:

The G4 strain was expanded and cultivated, the nematicidal active ingredients were extracted, and the nematicidal efficacy test was carried out to determine its effect on nematodes.

G4 cultivation method (the following are percentages by weight):

1. Test tube culture

The medium formula is: 1-3% yeast extract, 0.5-2% peptone; 1-4% glucose; 1.8-2% agar; pH6-7. Inoculate the G4 mycelium on the culture medium and culture it at 28-32°C for 1-4 days to obtain the test tube species;

2. Liquid expansion culture

The formula of the liquid medium is: 1-3% yeast powder; 10-20% soybean cake powder; 0.5-2% fish meal; 1-4 glucose; pH6-7, the rest is water. Inoculate the test tube seeds into 250ml Erlenmeyer flask liquid culture medium, each bottle contains 100ml, and culture on a shaking table at 25-35°C for 2-5 days with a rotation speed of 200-300rpm.

3. Extract nematode active ingredients

Centrifuge the fermentation culture at 8000rpm/min for 20 minutes, take the supernatant, add ammonium sulfate at a ratio of 1:0.85, let stand at 4°C for 2 hours, then centrifuge at 8000rpm/min for 30 minutes, and discard the supernatant. Redissolve with 50mM phosphate buffer (39ml of 50mM sodium dihydrogen phosphate plus 61ml of 50mM disodium hydrogen phosphate, pH 7), put the redissolved solution into a 21mm dialysis bag (MW: 8,000-14400KDa) at 10- Dialyze in 20 times of 50mM phosphate buffer solution for 3-4 times, each time for 3 hours, to obtain nematocidal liquid. Store the drug solution in a refrigerator at 4°C until use.

G4 nematicide activity test:

1. Preparation of test drugs

Cultivate the G4 strain according to the aforementioned liquid expansion culture method, and prepare the test agent according to the aforementioned method of extracting nematicide active ingredients.

2. Preparation of drugs for comparison

Control 1: Prepare the drug for the test by the above-mentioned 1 method for preparing the drug for the test, but the culture medium is not inoculated with the G4 strain.

Control 2: clean water was used as the control.

Control 3: In order to prove that the active ingredient of the nematode is protein, the prepared test agent was boiled and inactivated as a control.

2. Preparation of nematodes for experiment

(1) Preparation of Panagrellus redivivus nematode

P.redivivus nematodes were inoculated on oatmeal medium, cultured at 28°C for 6 days, and frozen at 4°C for later use. The required nematodes were washed out with the Behmann funnel method, placed in a 5ml centrifuge tube, washed with 5ml sterile water, centrifuged briefly, the supernatant was discarded, and repeated 5 times to obtain clean test nematodes. Dilute the nematodes with sterile water to a nematode suspension with a content of 15/μl.

(2) Preparation of pine wood nematode (Bursaphelenchus xylophilus)

Put 15g of corn kernels soaked in water for 2 days in a 100ml conical flask, add 10ml of water, autoclave, and insert Botrytis cinerea. Incubate at 25°C for 4 to 7 days. After the hyphae covered the Erlenmeyer flask, inoculate the pine xylophilus nematode sterilized by 0.25% sodium hypochlorite, and culture at 28° C. for 15 to 20 days. Wash the nematodes with sterile water to prepare a nematode suspension with a content of 15 nematodes/μl.

3. Test method:

(1) Drug efficacy test method

Take 200 μl of the test drug in a 1.5ml centrifuge tube, add 150 live nematodes, place the centrifuge tube flat, and place it at 25°C. , 48 hours, and 60 hours to check and calculate the mortality of nematodes. And observe under an optical microscope to observe the changes in the nematode body wall

The method for identifying death is: add 1-5 drops of 5% Nacl solution to the treatment centrifuge tube, observe after 2 minutes, dead worms are stiff, and live worms are curled or twisted.

Three kinds of control drugs were used respectively, and each treatment was repeated three times.

(2) Observation test of G4 decomposing nematodes

Live nematodes were treated with test agents at 25°C for 12 hours and 24 hours respectively, and the treated samples were washed three times with PBS buffer, fixed with 3% glutaraldehyde and acetic acid, and replaced with gradient glycerol and acetic acid. Ethyl ester was replaced again, vacuum freeze-dried, gold-coated, and finally fixed on a slide, observed with a scanning electron microscope (SEM). Treat and observe with the control drug at the same time.

4. Test results

Table 1 Insecticidal effect of G4 on Panagrellus redivivus nematode Pharmacy treatment Nematodes were fixed death situation 12 hours 24 hours 36 hours mortality rate nematode changes mortality rate nematode changes mortality rate nematode changes test drug 85 Insect rigidity 95 body wall dissolves 98 mostly dissolved Control 1 10 no change 20 undissolved 30 body wall intact Control 2 10 no change 10 undissolved 20 body wall intact Control 3 10 no change 10 undissolved 30 body wall intact

Table 2 Insecticidal effect of G4 on pine wood nematode Pharmacy treatment Nematodes were fixed death situation 24 hours 48 hours 60 hours mortality rate nematode changes mortality rate nematode changes mortality rate nematode changes test drug 80 Insect rigidity 95 Rough body wall, beginning to dissolve 96 partially dissolved Control 1 15 no change 20 undissolved 30 undissolved Control 2 5 no change 10 undissolved 15 undissolved Control 3 20 no change 20 undissolved 30 undissolved

The results show that the experimental medicament extracted from the liquid fermentation product of the G4 strain has good insecticidal effects on both Panagrellus redivivus nematodes and pine wood nematodes, and the average death rate of the nematodes is above 95%. Judging from the changes in the treatment of nematodes, it has gone through a process from partial body wall dissolution (Figure 3) to complete dissolution of the nematode body (Figure 1), indicating that the active ingredients are mainly enzymes that decompose nematodes. However, after boiling the test agents extracted from the same batch led to protein denaturation, the nematicidal activity was very small, and the nematode body wall did not appear to dissolve, which also proved that the active ingredients of G4 against nematodes were mainly enzymes.

The G4 strain of the present invention is obviously different from other Brevibacillus lateralsporosa producing parasporal crystals. Its main active ingredient is enzyme, and its toxicity to nematodes, especially pine xylophilus, is significantly higher than that of other similar microorganisms found so far. At the same time, the G4 bacterial strain grows extremely fast under the culture conditions of the present invention, and has good application and development prospects.

Description of drawings:

Figure 1 shows the decomposition of treated nematodes.

Figure 2 shows control nematodes with intact worm bodies.

Figure 3 shows the decomposition of the head of treated nematodes.

Figure 4 shows control nematodes with their heads kept intact.

Detailed ways:

The following are examples of the present invention, but the content of the present invention is not limited thereto.

Embodiment one:

Brevibacillus laterosporus G4 cells were inoculated on the slant of test tube agar medium, and the medium formula was: 2% yeast extract, 1.5% peptone; 2% glucose; 1.8% agar; pH7. The G4 mycelium was inoculated on the culture medium and cultured at 30° C. for 2 days to obtain test tube species.

Test tube seed is inoculated in 250ml Erlenmeyer flask liquid culture medium (every bottle packs 100ml), and liquid culture medium formula is: 2% yeast powder; 15% soybean meal; 1% fish meal; 3% glucose; pH7, all the other are water. The culture time was 3 days on a shaker at 30° C., and the rotation speed was 220 rpm.

Centrifuge the fermentation culture at 8000rpm/min for 20 minutes, take the supernatant, add ammonium sulfate at a ratio of 1:0.85, let stand at 4°C for 2 hours, then centrifuge at 8000rpm/min for 30 minutes, and discard the supernatant. Redissolve with 50mM phosphate buffer (39ml of 50mM sodium dihydrogen phosphate plus 61ml of 50mM disodium hydrogen phosphate, pH 7), put the redissolved solution into a 21mm dialysis bag (MW: 8,000-14400KDa) at 10- Dialyze in 20 times of 50mM phosphate buffer solution for 3-4 times, each time for 3 hours, to obtain nematocidal liquid.

Embodiment two:

It is basically the same as Example 1, except that the formula of the liquid medium is: 3% yeast powder; 10% soybean meal; 2% fish meal; 1% glucose.

Embodiment three:

It is basically the same as Example 1, except that the formula of the liquid medium is: 1% yeast powder; 20% soybean meal; 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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