CN1279160C - Acremonium elegans bacterial agent with nematodeicidal function and its preparation method and application - Google Patents

Abstract

The present invention relates to a bactericidal monacrosporium elegans agent with a wireworm killing function and a preparation method and an application thereof, which belongs to the technical field of microbial pesticides. The production strain of the bactericidal agent is monacrosporium elegans, and the number of the production strain is YMF1.00027. The bactericidal agent is prepared by a conventional method, wherein a liquid fermentation culturing medium has the formula of 0.1 % of glucose, 0.1% of ammonium sulfate, 0.05% of magnesium sulfate, 100g of potato boiling juice and 0.001% of ferrous sulfate heptahydrate, wherein water is added to the formula until the volume is 1L and the pH value is 6.5. The method for extracting the effective ingredients of wireworms from a liquid fermentation product comprises the following steps: a fermentation culturing product is filtered, and supernatant fluid concentrated by the ammonium sulfate can be used as medicine liquor for killing the wireworms. The bactericidal agent has high toxicity to panagrellus redivivus wireworms and pine wireworms, and has good application prospects.

Description

Acremonium elegans bacterial agent with nematodeicidal function and its preparation method and application

Technical field:

The invention relates to a Caenorhamona elegans fungus agent with a nematicide function, a preparation method and application thereof, and belongs 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 global losses caused by nematodes are as high as 100 billion U.S. dollars every year. 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. Invented in the 1940s, chemical nematicides have played an important role in ensuring agricultural production and preventing nematode diseases for a long time. 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 use of methyl bromide and methylene bromide has been banned in the United States. Chemical nematicides have brought serious negative impacts on human health and the environment while preventing nematodes and ensuring agricultural production. It is imminent to develop high-efficiency and low-toxic nematicides and biological nematicides.

Monocremonium is a very important nematode-predating fungus, which can produce three-dimensional fungus nets and other predatory organs to catch nematodes, and is a very important nematode biocontrol resource. Studies have shown that Caenorhamona elegans can not only produce traps to catch nematodes on the CMA plate, but also secrete extracellular proteases, which can decompose the nematode body wall by using extracellular proteases to kill nematodes.

Invention content:

The purpose of the present invention is to develop a biological nematicide through the research on Caenormona elegans which produces extracellular enzymes and can effectively decompose the body wall of nematodes.

The Monacrosporium elegans (Monacrosporium elegans), YMF1.00027 and YMF1.00027 strains screened by the present invention, which have a good poisonous effect on plant parasitic nematodes, have been preserved in the China Committee for the Preservation of Microorganisms on January 27, 2005. General Microbiology Center; Address: Zhongguancun, Beijing, China; the number of the preservation registration is CGMCC No.1310.

Monacrosporium elegans, YMF1.00027: colorless vegetative hyphae, separate, branched; on CMA medium, at 28°C, the growth rate is fast, and the colony is colorless; the length of the conidiophores is 120-575 μm , separated, upright, sometimes long branches, single conidia at the top; conidia spindle-shaped, 37.5-72.5×17.5-27.5μm in size, 1-4 septa, mostly three septa, extra large in the center The cells are obvious, and the bacteria can also produce microconidia, obovate, 8-15×5-6μm in size, without septa. The prey organ is a three-dimensional bacterial net.

The present invention is prepared according to a conventional method. After expanding and culturing Monacrosporium elegans (Monacrosporium elegans) YMF1.00027, the nematicidal active ingredient is extracted, and its poisonous effect on saprophytic nematodes (Panagrellus redivivus) and pine wood nematodes is determined.

The present invention is achieved like this:

YMF1.00027 strain culture method:

1. Test tube culture

The medium formula is improved PDA medium: 1% glucose; 100g potato juice, 1.8% agar; pH 6.5. The YMF1.00027 mycelium was inoculated on the culture medium and cultured at 22°C for 8 days to obtain test tube species.

2. Liquid expansion culture (enzyme production culture)

The composition of enzyme production medium (PL-4) is as follows: 0.1% glucose, 0.1% ammonium sulfate, 0.05% magnesium sulfate, 100g potato juice, 0.001% ferrous sulfate, supplemented with water to 1L, pH 6.5. Each 250ml Erlenmeyer flask was divided into 60ml of culture solution, and a small amount of nematode was added as an inducer, sterilized at 121°C for 20 minutes, inserted into the bacterial block, and cultured on a shaker (200rpm) at 26°C for 6 days.

Extraction of nematicidal active ingredients:

Filter the fermentation culture, collect the supernatant, add ammonium sulfate at a ratio of 1:0.56, let stand at 4° C. for 2 hours, then centrifuge at 8000 rpm/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 7.0), put the redissolved solution into a 21mm dialysis bag (MW: 8,000-14400KDa) in 50mM phosphoric acid Dialyze in the buffer solution 3-4 times, each time for 3 hours, to obtain the nematocidal liquid. Store the drug solution in a refrigerator at 4°C until use.

Nematicidal Activity Test:

1. Preparation of the test drug and the control drug

1) Cultivate the YMF1.00027 strain according to the above-mentioned liquid expansion culture method, and prepare the test agent according to the above-mentioned method of extracting nematicide 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 YMF1.00027 strain is not inserted into the culture solution.

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 experiment

1) Inoculate P.redivivus nematodes on oatmeal medium, culture them at 28°C for 6 days, and freeze them in a refrigerator 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) 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 and dilute to a nematode suspension with a content of 15/μl.

3. Test method

1) Drug efficacy test method

Take 200 μl of the test drug in a 1.5ml centrifuge tube, add 60 nematodes of Panagrellus redivivus and pine xylophilus respectively, place the centrifuge tubes flat, place them at 25°C, and check and calculate the death of nematodes at 12 hours, 24 hours and 36 hours respectively Rate. 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 control agents were used respectively, and each treatment was repeated three times.

Control experiments were carried out with two control agents respectively.

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

4. Test results

Table 1 Toxic effect of Caenormona elegans YMF1.00027 on nematode Panagrellus redivivus Pharmacy treatment Nematode immobilization and death 12 hours 24 hours 36 hours mortality rate(%) nematode changes mortality rate(%) nematode changes mortality rate(%) nematode changes test drug 70 Partially degraded 90 Partially degraded 100 Completely degraded Control 1 10 body wall intact 10 body wall intact 10 body wall intact Control 2 10 body wall intact 15 body wall intact 15 body wall intact

Table 2 The poisonous effect of Caenormona elegans YMF1.00027 on pine wood nematode Pharmacy treatment Nematode immobilization and death 12 hours 24 hours 36 hours mortality rate(%) nematode changes mortality rate(%) nematode changes mortality rate(%) nematode changes test drug 60 Insect rigidity 70 Insect rigidity 85 Insect rigidity Control 1 5 body wall intact 10 body wall intact 10 body wall intact Control 2 5 body wall intact 10 body wall intact 15 body wall intact

The results showed that the test agent extracted from the liquid fermentation product of Monacrosporium elegans YMF1.00027 had a good poisonous effect on Panagrellus redivivus nematodes, and the nematode mortality rate reached 100% within 36 hours, and the body wall was completely dissolved. Judging from the changes in the treatment of nematodes, it has gone through the process from partial body wall dissolution to complete dissolution of the nematodes, indicating that the active ingredients are mainly enzymes that decompose nematodes. However, after boiling the test agents extracted from the same batch to cause protein denaturation, the nematicidal activity is very small, and the nematode body wall does not appear to dissolve. It shows that the active ingredients of Caenormona elegans YMF1.00027 strain are mainly enzymes. Monacrosporium elegans (Monacrosporium elegans) YMF1.00027 strain has a certain poisonous effect on pine wood nematodes, and the nematode mortality rate reaches 85% within 36 hours, and the worm body is rigid.

The C. acremonium fungus agent of the invention has good poisonous effect on the nematode Panagrellus redivivus and the pine wood nematode, and can be developed as a nematode biocontrol agent.

Detailed ways:

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

The YMF1.00027 strain was inoculated on the modified PDA slant, and the medium formula was: 1% glucose; 100g potato boiled juice, 1.8% agar; the pH was natural. The YMF1.00024 mycelium was inoculated on the culture medium and cultured at 22°C for 8 days to obtain test tube species.

The test tube seed is inoculated in the 250ml Erlenmeyer flask liquid medium (60ml of every bottle), and the liquid medium formula is: 0.1% glucose, 0.1% ammonium sulfate, 0.05% magnesium sulfate, 100g potatoes are boiled into juice, 0.001% ferrous sulfate, Make up to 1 L with water, pH 6.5. Each 250ml Erlenmeyer flask was filled with 60ml of culture solution, and a small amount of nematodes were added as an inducer, sterilized at 121°C for 20 minutes, inserted into the bacterial block, and cultured on a shaker (200rpm) at 26°C for 6 days.

Add ammonium sulfate at a ratio of 1:0.56, let stand at 4°C for 2 hours, then centrifuge at 8000 rpm/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 7.0), put the redissolved solution into a 21mm dialysis bag (MW: 8,000-14400KDa) in 50mM phosphoric acid Dialyze in the buffer solution 3-4 times, each time for 3 hours, to obtain the nematocidal liquid. Store the drug solution in a refrigerator at 4°C until use.

The use effect of the present invention is shown in Table 1 and Table 2.

Claims (3)

1, a kind of Caenorhabdina elegans fungal agent with nematicide function, it is characterized in that: (1) The production strain is Monacrosporium elegans YMF1.00027, and the preservation registration number is CGMCC No.1310; (2) The Caenorhamona elegans agent is obtained through the following steps: a. Test tube culture Inoculate YMF1.00027 mycelia to 1% glucose; 100g potato juice, 1.8% agar; pH6.5 medium, culture at 22°C for 8 days to obtain test tube species; b. Liquid expansion culture Each 250ml Erlenmeyer flask was divided into 60ml of culture solution, and a small amount of nematode was added as an inducer, sterilized at 121°C for 20 minutes, inserted into the test tube, and then cultured at 26°C in a shaker at 200rpm for 6 days; The solution is: 0.1% glucose, 0.1% ammonium sulfate, 0.05% magnesium sulfate, 100g potatoes boiled into juice, 0.001% ferrous sulfate, add water to 1L, pH 6.5; c. Extraction of nematicide active ingredients Filtrate the fermentation culture, collect the supernatant, add ammonium sulfate at a ratio of 1:0.56, let stand at 4°C for 2 hours, then centrifuge at 8000rpm/min for 30 minutes, discard the supernatant; redissolve with 50mM phosphate buffer, The 50mM phosphate buffer solution is: 39ml of 50mM sodium dihydrogen phosphate plus 61ml of 50mM disodium hydrogen phosphate, the pH is 7.0; the solution obtained by redissolving is put into a 21mm dialysis bag with MW: 8,000-14400KDa, and dissolved in 50mM phosphate buffer Dialyze 3-4 times, each time for 3 hours, to obtain the nematicidal medicinal solution, and store the medicinal solution in a refrigerator at 4°C until use. 2. The preparation method of the Acremonium cerevisiae fungal agent according to claim 1, comprising three steps of test-tube seed cultivation, liquid expansion cultivation, and extraction of nematicide active components, characterized in that: a. Test tube culture Inoculate YMF1.00027 mycelia to 1% glucose; 100g potato juice, 1.8% agar; pH6.5 medium, culture at 22°C for 8 days to obtain test tube species; b. Liquid expansion culture Each 250ml Erlenmeyer flask was divided into 60ml of culture solution, and a small amount of nematode was added as an inducer, sterilized at 121°C for 20 minutes, inserted into the test tube, and then cultured at 26°C in a shaker at 200rpm for 6 days; The solution is: 0.1% glucose, 0.1% ammonium sulfate, 0.05% magnesium sulfate, 100g potatoes boiled into juice, 0.001% ferrous sulfate, add water to 1L, pH 6.5; c. Extraction of nematicide active ingredients Filtrate the fermentation culture, collect the supernatant, add ammonium sulfate at a ratio of 1:0.56, let stand at 4°C for 2 hours, then centrifuge at 8000rpm/min for 30 minutes, discard the supernatant; redissolve with 50mM phosphate buffer, The 50mM phosphate buffer solution is: 39ml of 50mM sodium dihydrogen phosphate plus 61ml of 50mM disodium hydrogen phosphate, the pH is 7.0; the solution obtained by redissolving is put into a 21mm dialysis bag with MW: 8,000-14400KDa, and dissolved in 50mM phosphate buffer Dialyze 3-4 times, each time for 3 hours, to obtain the nematicidal medicinal solution, and store the medicinal solution in a refrigerator at 4°C until use. 3. The application of the C. elegans bacterial agent as claimed in claim 1, characterized in that the bacterial agent is used for biocontrol of Panagrellus redivivus nematode and pine wood nematode.