CN1468523A - A kind of biological toxin for controlling pine wood nematode and its preparation method - Google Patents

Abstract

The invention relates to a biological toxin for preventing and treating pine wood nematodes and a preparation method, belonging to the technical field of biological pesticides. The production strain Pseudohalonectria adversaria (ZD1) of the biotoxin has been preserved in the General Microorganism Center of China Microorganism Culture Collection Management Committee on May 26, 2003, and the preservation number is CGMCC NO.0931. After ZD1 is cultivated in liquid and grain solid, effective water-soluble and fat-soluble substances are extracted from metabolites to prepare nematicidal biotoxins. The invention has good poisonous effect on pine xylophilus, and has good application and development prospects.

Description

A kind of biological toxin for controlling pine wood nematode and its preparation method

Technical field:

The invention relates to a biological toxin for preventing and treating pine wood nematodes and a preparation method, belonging to the technical field of biological pesticides

Background technique:

Pine wood nematode (Bursaphelenchus xylophilus) disease is a catastrophic catastrophe to pine species, and it is an important quarantine object in the world. Since the first discovery of pine wood nematode disease in Nanjing Zhongshanling forest area in 1982, it has spread in Jiangsu, Anhui, Guangdong, Zhejiang, Shandong, Taiwan and Hong Kong and other provinces and regions. The landscape is severely damaged and poses a serious threat to pine forests in a wide range of suitable habitats. Because there is no effective prevention and treatment agent in the world at present, the disease is also called "pine tree cancer". As the disease has approached Huangchu Scenic Area, in order to protect the wonders of Huangshan, the state and Anhui Province invested nearly 60 million yuan to establish the "Huangshan Pine Wood Nematode Prevention System Project". After the outbreak of the disease in Guangdong Province, the quarantine control was neglected for a time, resulting in further expansion and spread.

At present, there are no effective pesticides and control methods for pine wood nematode at home and abroad. In general, conservative control methods are adopted after the occurrence of pine wood nematode, that is, the susceptible pine trees are cut down and burned to cut off the transmission. Biological agents that are effective against plant-parasitic root-knot and cyst nematodes are not active at all against B. xylophilus. It is imminent to find effective control methods and agents for pine xylophilus.

Invention content:

The purpose of the present invention is to select fungal strains with good insecticidal activity against pine wood nematodes. After the bacterial strains are cultured by conventional liquid fermentation and wheat grain solid fermentation of the present invention, the effective ingredients for killing pine wood nematodes are extracted from metabolites to prepare biotoxins. , in order to find effective control agents and methods for pine wood nematode.

The fungus used in the present invention is Pseudohalonectria adversaria (ZD1), depository unit: China Microbiological Strain Preservation Management Committee General Microbiology Center; address: China. Beijing. Zhongguancun; preservation date: May 26, 2003; preservation registration number CGMCC NO.0931. P. adversaria (ZD1) belongs to aquatic fungi, and is a strain with good nematicidal activity against pine xylophilus selected by the inventor from a large number of aquatic fungal strains.

The present inventor found that fungi of the genus Pseudohalonectria have good insecticidal effects on pine wood nematodes through research, and screened and determined a strain Pseudohalonectria adversaria (ZD1) with the best properties from the genus Pseudohalonectria for further research. In the invention, the ZD1 bacterial strain is subjected to liquid fermentation and barley solid fermentation, and then extracts metabolites with activity of killing pine wood nematodes to prepare biological nematode toxins.

Fungus P.adversaria (ZD1) culture method of the present invention (below is weight percent):

The liquid medium formula is 1-4% soybean; 1-5% glucose; 1-2% starch; 0.1-0.5% yeast extract; 0.2-1% sodium chloride; 0.02-0.1% potassium dihydrogen phosphate; 0.02- 0.1% magnesium sulfate; 0.1-0.6% calcium carbonate, the rest is water.

The formula of wheat grain solid medium is 0.2-1% calcium carbonate; 1-3% soybean flour; 0.2-1% ammonium sulfate; 0.5-3% calcium superphosphate; wheat grain 92%-96%.

The invention is divided into two methods for extracting water-soluble nematicidal biotoxin by liquid fermentation and extracting fat-soluble nematicidal biotoxin by solid fermentation of wheat grains.

Preparation method for extracting nematicide biotoxin by liquid fermentation:

1. Inoculate the mycelium of ZD1 on the slant of the test tube agar medium, the medium formula is PDA medium, and cultivate it at 18-26°C for 6-12 days to obtain the test tube species;

2. Inoculate the test tube seed into a 250ml Erlenmeyer flask (70ml per bottle) liquid medium, the medium formula is the above-mentioned liquid medium formula, cultivate on a shaker at 20-30°C, the rotation speed is 200-300rpm, and the culture time is 5- 8 days.

3. Put the fermented liquid containing mycelium into a centrifuge, centrifuge at 3600 rpm for 20 minutes, and filter to obtain a water-soluble biotoxin that kills pine wood nematodes.

Preparation method for extracting pine xylophilus biotoxin by solid fermentation of wheat grains:

1. The test-tube seed culture method is the same as the aforementioned liquid fermentation production method 1;

2. Adopt the formula of the aforementioned wheat grain solid medium. After soaking the wheat in clean water for 24 hours, pick it up and put it in boiling water to cook. It is suitable for the skin to be cooked but not broken. Pick up the filtered water and mix it with auxiliary materials and put it into a 1000ml Erlenmeyer flask, 700ml per bottle. After sterilizing at 120°C for 30 minutes, put it into a test tube and cultivate it at 22-26°C for 15-20 days until the hyphae are full.

3. Dig out the wheat grains covered with hyphae, freeze-dry, use chloroform and methanol in a ratio of 1:1 to make solvent extraction, concentrate after extraction, and obtain the total crude extract. The crude extract is dissolved in a small amount of dimethyl sulfoxide (DMSO), and then dissolved in a certain concentration of Tween-20 (Tween-20) solution to obtain a fat-soluble emulsion of biotoxin for killing pine wood nematodes. In the emulsion, the final concentration of dimethyl sulfoxide does not exceed 3%, and the final concentration of Tween-20 does not exceed 0.3%.

Water-soluble nematicidal biotoxins are mainly extracted by liquid fermentation culture, while fat-soluble nematicidal biotoxins are mainly extracted by solid fermentation of wheat grains.

Drug efficacy test of product of the present invention to pine xylophilus

Drug efficacy test 1:

1. Test agent: produce nematicide biotoxin according to the liquid culture method; use uninoculated culture medium after centrifugation at 3600 rpm for 20 minutes and then filter as a control.

2. Pine wood nematode preparation: put 15 g of corn kernels soaked in water for 2 days in a 100 ml triangular flask, add 10 ml 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: take a petri dish with a diameter of 6 cm, add 2ml of the test agent after sterilization, then add 20 μl of nematode suspension (300 pieces), place at 25°C for cultivation, regularly check the life and death of nematodes with a dissecting microscope, under physiological stimulation Nematode non-response was determined as nematode death, and the efficacy of the bionematicidal toxin was calculated based on the mortality rate.

The extract of the uninoculated culture medium was used as the control, and each treatment was repeated three times.

4. Test results

Table 1 Insecticidal effect of liquid fermentation biotoxins on pine xylophilus deal with 12 hours 24 hours 48 hours Number of dead insects mortality rate(%) Drug efficacy (%) Number of dead insects mortality rate(%) Drug efficacy (%) Number of dead insects mortality rate(%) Drug efficacy (%) potion 1 245 81.6 81.3 257 85.6 85.6 262 87.3 87 potion 2 244 81.3 81 254 84.6 84.3 257 85.6 85.3 potion 3 209 69.6 69.3 258 86 85.6 268 89.3 89 average 233 77.5 77.2 256 85.4 85 262 87.4 87.1 control 1 1 1

The result shows: the product water-soluble biological nematicide toxin of the present invention has good desinsection effect to pine xylophilus, and its 12-hour average mortality rate reaches 77.5%, reaches 81.6% at most; 48 hours average mortality rate reaches 87.4%, reaches 89.3% %, the 48-hour average efficacy reached 87.1%.

Drug efficacy test 2:

1. Drugs for the test: the formula of wheat grain solid medium is 1% calcium carbonate; 3% soybean powder; 0.5% ammonium sulfate; 0.5% superphosphate; 95% wheat grain. The test agent was prepared according to the above-mentioned preparation method of extracting pine wood nematode biotoxin by solid fermentation of wheat grains.

Control drug: according to the above-mentioned wheat grain solid fermentation production method, but the test tube species is not inserted into the wheat grain solid medium.

2. The preparation of pine wood nematode is the same as that of drug efficacy test 1.

3. The test method is the same as the efficacy test 1.

4. Test results

Table 2 Insecticidal effect of solid fermented biotoxins on pine wood nematode deal with 12 hours 24 hours 36 hours Number of dead insects mortality rate(%) Drug efficacy (%) Number of dead insects mortality rate(%) Drug efficacy (%) Number of dead insects mortality rate(%) Drug efficacy (%) Pharmacy (average) 16 5.3 5.3 27 9 9 294 98 98 control 0 0 0

The results show that the fat-soluble biotoxin of the present invention has a good insecticidal effect on pine wood nematodes. Compared with water-soluble substances, its drug effect is slower, and the drug effect is not significant at 12 hours and 24 hours, and the drug effect is remarkable at 36 hours And more than water-soluble biological toxins, the mortality rate reaches 98%, showing its good application and development prospects.

Detailed ways:

Embodiment one:

Inoculate the mycelium of Pseudohalonectria adversaria ZD1 on the slant of the test tube agar medium, the medium formula is PDA medium, and cultivate it at 20°C for 9 days to obtain the test tube species.

Test tube seed is inoculated in (every bottle 70ml) liquid medium in the 250ml Erlenmeyer flask again, and medium formula is 2% soybean; 2% glucose; 0.5% starch; 0.2% yeast extract; 0.4% sodium chloride; 0.05% Potassium dihydrogen phosphate; 0.05% magnesium sulfate; 0.2% calcium carbonate, and the rest is water; cultured on a shaking table at 22° C. with a rotation speed of 250 rpm for 8 days.

Then put the fermented liquid containing mycelia into a centrifuge for centrifugation at a speed of 3600 rpm for 20 minutes, and filter to obtain a water-soluble biotoxin that kills pine wood nematodes.

Embodiment two:

The test tube culture method is the same as in Example 1.

The test tube seed is inoculated on the wheat grain solid medium, and the medium formula is 1% calcium carbonate; 3% soybean powder; 0.5% ammonium sulfate; 0.5% superphosphate; and 95% wheat grain. Soak the wheat grains in clean water for 24 hours, pick them up and put them in boiling water to cook. It is better if they are cooked but the skin is not broken. Pick up the water and mix them with auxiliary materials and put them into a 1000ml triangular flask. Each bottle is 700ml. After sterilizing at 120°C for 30 minutes, culture at 22°C for 20 days after inoculation.

Then dig out the solid-cultured bacterial strain, freeze-dry, use chloroform and methanol in a ratio of 1:1 to prepare a solvent for leaching, and concentrate after leaching to obtain the total crude extract. The crude extract is dissolved in a small amount of dimethyl sulfoxide (DMSO), and then dissolved in a certain concentration of Tween-20 (Tween-20) solution to obtain the emulsion of the biological preparation for killing pine wood nematodes. In the emulsion, the final concentration of dimethyl sulfoxide does not exceed 3%, and the final concentration of Tween-20 does not exceed 0.3%.

Embodiment three:

Basically the same as Example 2, the difference is the formula of the medium, which is 0.5% calcium carbonate; 1% soybean powder; 0.5% ammonium sulfate; 2% superphosphate; 96% wheat grains.

Embodiment four:

Basically the same as Example 2, the difference is the medium formula, which is 1% calcium carbonate; 3% soybean flour; 1% ammonium sulfate; 3% superphosphate; 92% wheat grains.

Claims (2)

1. A biotoxin for preventing and controlling pine wood nematode, which is prepared by extracting and preparing production strains and auxiliary materials by culturing liquid and grain solids, and is characterized in that the production strain of the biotoxin is Pseudohalonectria adversaria ZD1, and the ZD1 strain was produced in May 2003 On the 26th, it was deposited in the General Microbiology Center of China Microbiological Culture Collection Management Committee, and the preservation number is CGMCC NO.0931. 2. A method for preparing a biotoxin for preventing and treating pine wood nematode, prepared by liquid fermentation and solid fermentation, characterized in that: 2.1 The formula of wheat grain solid medium is: 0.2-1% calcium carbonate, 1-3% soybean powder, 0.2-1% ammonium sulfate, 0.5-3% superphosphate, 92-96% wheat grain; 2.2 The method for preparing the biological nematicide toxin by solid fermentation of wheat grains is to dig out the wheat grains covered with mycelia and freeze-dry; use chloroform and methanol in a ratio of 1:1 to make solvent extraction, concentrate after extraction, and obtain The total crude extract, the crude extract is dissolved in a small amount of dimethyl sulfoxide, and then dissolved with a certain concentration of Tween-20 solution to obtain an emulsion of biological preparations for killing pine wood nematodes; in this emulsion, dimethyl sulfoxide The final concentration of sulfone does not exceed 3%, and the final concentration of Tween-20 does not exceed 0.3%.