CN1663394A - A kind of nematicide biological agent and its preparation method 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 nematicide biological agent and its preparation method and application

Technical field:

The invention relates to a nematicide biological bacterial agent and its preparation method and application, belonging to the technical field of biological pesticides.

Background technique:

Plant nematodes are a common plant disease worldwide. There are more than 70 known species of root-knot nematodes alone, which harm more than 3,000 plants and cause huge losses every year. According to the statistics of the Food and Agriculture Organization of the United Nations, the annual global losses caused by nematodes are as high as more than 100 billion US dollars. In 2001, the losses caused by nematode damage to crops in my country were as high as 23.3 billion 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. What's more serious is that root-knot nematodes cause crop wounds after infestation, resulting in serious root diseases of crops.

At present, chemical nematicides are still the main method of nematode control. For a long time, chemical nematicides have played an important role in ensuring agricultural production and preventing nematode diseases. However, with the advancement of science and technology, it was found that many chemical nematicides have side effects, which not only produce drug resistance, but also cause environmental pollution and endanger human health. Many of them have been banned or will be banned one after another. For example, the United States has banned the use of methyl bromide and methylene bromide. While chemical nematicides prevent nematodes and ensure agricultural production, they also have negative impacts on human health and the environment. It is urgent to develop high-efficiency and low-toxicity nematicides and biological nematicides.

Verticillium is a very important class of biocontrol fungi, many of which are important pathogenic fungi of insects and nematodes, can effectively control a variety of harmful insects and nematodes, and play a role in the biological control of plant pathogenic insects and nematodes very important role. Among them, Verticillium lecanii and Prokonia clastica have been used in the development of bio-insecticides, but there is no report about the effect of Verticillium canespora on insects and nematodes.

Invention content:

The purpose of the present invention is to develop a nematicidal biological agent through the research of an endoparasitic fungus Verticillium ciferium which produces extracellular enzymes and can effectively decompose the nematode body wall structure.

The inventors screened an endoparasitic fungus Lecanicillium psalliotae, YMF1.00112, which has a good poisoning effect on plant parasitic nematodes. The YMF1.00112 strain has been preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee; address: China. Beijing. Zhongguancun; preservation date: January 27, 2005; preservation registration number CGMCC No.1312.

Lecanicillium psalliotae, YMF1.00112 is an endoparasitic fungus isolated from nematode body surface by the inventor, the main morphological characteristics are:

It grows faster on PDA medium. After 7 days of culture, the diameter of the colony reaches 3-6cm. The mycelium grows densely. The color of the colony is white, and the back is light yellow to brown. 0.2-0.5 μm wide, conidia head-shaped, oval, sickle-shaped, 0.6-2.2×2.0-9.0 μm, no chlamydospores.

The present invention adopts the conventional method to expand the culture of Verticillium ciferium, extracts nematicidal active ingredients, and measures its insecticidal effect on saprophytic nematodes (Panagrellus redivivus) and pine wood nematodes (Bursaphelenchus xylophilus).

The present invention is achieved like this:

YMF1.00112 strain culture method:

1. Test tube culture

The test-tube species is cultivated by a conventional method, inoculating the YMF1.00112 mycelium on the PDA medium, and culturing at 20-25°C for 7-10 days to obtain the test-tube species.

2. Liquid expansion culture

The formula of the liquid medium is: 8-15g glucose; 0.8-1.5g ammonium sulfate; 0.4-0.6g magnesium sulfate; 0.01-0.02g ferrous sulfate; 1000ml potato extract juice (100g potato plus 1200ml water boiled and filtered, water to make up to 1000ml ), pH6.5. Use 250ml Erlenmeyer flasks, each filled with 60ml of culture solution, sterilized at 121°C for 20 minutes, then insert into test tubes, and culture on a shaker at 26°C, 200rpm for 6 days.

Extraction of nematicidal active ingredients:

After the bacteria solution that the aforementioned liquid has been expanded and cultivated is filtered, it is concentrated with an ultrafiltration membrane with a pore size of 5KDa. After dialysis treatment, the concentrate was purified with a cation exchange column, and the breakthrough peak and elution peak were collected separately. The biological activity measurement showed that most of the protease activity was in the elution peak, and the combined elution peak was further purified with a hydrophobic column, and the elution peak was collected. The peak was dialyzed with distilled water, and electrophoresis analysis had reached electrophoretic purity. Freeze-drying: Freeze-dry the sample and dissolve it in 2ml of distilled water, store at -20°C, and the preparation after being concentrated by ultrafiltration membrane is the nematicide preparation.

Nematicidal Activity Test:

1. Preparation of the test drug and the control drug

1) Preparation of test agents

The bacterial liquid was obtained according to the aforementioned YMF1.00112 bacterial strain culture method, and the experimental medicament was prepared according to the aforementioned method of extracting nematicidal active ingredients.

2) Preparation of pharmaceuticals for comparison

Control 1: Prepared according to the method of preparing the test drug, the difference is that the YMF1.00112 strain is not inserted into the culture medium;

Control 2: The prepared test agent was boiled at 100° C. for 30 minutes as a control to prove that the nematicidal active ingredient is protease.

2. Preparation of Nematodes for Experiments

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 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) Prepare 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 60 live nematodes, place the centrifuge tube flat, and place it at 25°C. Check and calculate the mortality of nematodes. 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.

Control experiments were carried out with 2 kinds of control drugs respectively.

The experiment was set up in three parallels and repeated twice.

2) Nematicidal effect of the purified sample and decomposition of the nematode body wall: the experimental method is the same as above, and the decomposition effect of the purified sample on the nematode body wall is observed with an optical microscope.

4. Test results

Table 1: Insecticidal effect of YMF1.001 12 on Panagrellus redivivus nematode

Table 2: Insecticidal effect of YMF1.00112 on pine wood nematode (Bursaphelenchus xylophilus)

The results showed that the test agents extracted from the liquid fermentation product of YMF1.00112 strain had good nematicidal effects on both Panagrellus redivivus nematodes and pine wood nematodes. Treat nematodes with 5-fold concentrated samples as liquid medicine, 100% of Panagrellus redivivus nematodes are dead and the nematodes are completely decomposed after 12 hours, and the death rate of pine wood nematodes also reaches more than 60% (Table 2), and the death of pine xylophilus after 20 hours The rate also reached 100%, but the body wall structure of pine xylophilus was intact and not decomposed. The experimental results also showed that the nematicide activity of the sample purified by the cationic column was lower than that of the crude concentrated sample, indicating that other components were involved in the nematode killing process.

Judging from the changes in the treatment of nematodes, it has experienced a process from partial body wall decomposition to complete degradation of the nematodes, indicating that the active ingredients are mainly enzymes that decompose nematodes. However, the nematicidal activity of the test agents extracted from the same batch is very small after boiling to denature the protein, and the nematode body wall does not appear to dissolve, which also proves that the active ingredient of the YMF1.00112 strain against nematodes is mainly the enzyme kind.

Verticillium cannifria YMF1.00112 of the present invention is a kind of fungus that has good poisonous function to nematodes, and its main active ingredient is extracellular enzyme, and the toxicity to nematodes especially pine xylophilus is significantly higher than or equal to The previously reported P. chlamydosporum has a good prospect for application and development.

Detailed ways:

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

Embodiment one:

The YMF1.00112 strain was inoculated on a PDA slant, and cultured at 20-25°C for 8 days to obtain a test tube species.

Inoculate the test tube seed into 250ml Erlenmeyer flask liquid culture medium, each bottle contains 60ml, the liquid medium formula is: 10g glucose, 1g ammonium sulfate, 0.5g magnesium sulfate, 0.015g ferrous sulfate, 1000ml potato extract juice, pH6.5. Each 250ml Erlenmeyer flask was filled with 60ml of culture solution, sterilized at 121°C for 20 minutes, then inserted into the test tube, cultured on a shaker at 26°C and 200rpm for 6 days, then filtered the bacterial solution, and concentrated it with an ultrafiltration membrane with a pore size of 5KDa. After dialysis treatment, the concentrate was purified with a cation exchange column, and the breakthrough peak and elution peak were collected separately. The bioactivity measurement showed that most of the protease activity was in the elution peak, and the combined elution peak was further purified with a hydrophobic column, and the elution peak was collected. The peak was dialyzed with distilled water, and electrophoresis analysis had reached electrophoretic purity. Freeze-drying Samples were freeze-dried and dissolved in 2 ml of distilled water and stored at -20°C. The preparation concentrated by the ultrafiltration membrane is the nematicide preparation.

Embodiment two:

Basically the same as Example 1, the difference is: the formula of the liquid culture medium is 8g of glucose; 1.5g of ammonium sulfate; 0.6g of magnesium sulfate; 0.01g of ferrous sulfate.

Embodiment three:

Basically the same as Example 1, the difference is: the formula of liquid culture medium is: 15g glucose; 0.8g ammonium sulfate; 0.4g magnesium sulfate; 0.02g ferrous sulfate.

Use effect 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.