CN1288239C - Nematode-eating fungus with nematode-killing function, preparing method and use thereof - Google Patents

Abstract

The invention relates to a nematode-eating fungus with the function of killing nematodes and its preparation method and application, belonging to the technical field of microbial pesticides. After the bacterial strain of the present invention undergoes liquid fermentation, the nematicidal active ingredient is extracted from metabolites to prepare the nematode-eating fungus. The production strain is Dactylella shizishanna YMF1.00022, and its liquid fermentation medium formula is: 0.1-0.2% gelatin; 0.6-0.8% tryptone; 0.1-0.2% yeast extract powder; 0.05% ammonium sulfate; 0.001% ferrous sulfate; 0.05% magnesium sulfate; 1.129% disodium hydrogen phosphate; 1.238% sodium dihydrogen phosphate pH6.5, the rest is water. The method for extracting the nematicidal active ingredient is to take the supernatant of the fermentation culture, slowly add ammonium sulfate at a ratio of 1:0.56, let stand at 4° C. for 2 hours, then centrifuge at 8500 rpm for 15 minutes, and discard the supernatant. Re-dissolve with 10mM phosphate buffer, put the re-dissolved solution into a 21mm dialysis bag, and dialyze 3-4 times in 10-20 times of 10mM phosphate buffer, 3 hours each time. The product of the invention has the outstanding advantage of high toxicity to nematodes and has good potential application prospects.

Description

A nematode-eating fungus with nematicidal function and its preparation method and application

Technical field:

The invention relates to a nematode-eating fungus with the function of killing nematodes and its preparation method and application, belonging 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. For a long time, nematode control has mainly relied on chemical control. Although chemical nematicides have played an important role in nematode control, 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 of them have been banned or will be banned 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.

Nematode-eating fungi are a type of natural enemy of nematodes, and nematode biocontrol agents that are successfully launched on the market are all derived from such fungi. Among them, Aldactylum is an important group.

Invention content:

The purpose of the present invention is to develop a biological nematicide through the research of a strain of Dymphagium shizishan which produces extracellular protease and decomposes the body wall of nematodes.

In the study of nematode fungi carried out by the present inventors, a new species of the genus Septigmatinus, which has a very good poisonous effect on nematodes, was isolated from a large number of samples and named as Shizishan Seperodactylus. Dactylellashizishanna YMF1.00022, the YMF1.00022 strain has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee; address: Zhongguancun, Beijing, China; preservation date: January 27, 2005; preservation registration number CGMCC No. 1311.

Dactylella shizishanna (Dactylella shizishanna) YMF1.00022 bacterial strain morphological characteristics of the present invention are: bacterial colony is nearly colorless on CMA medium, and aerial hyphae are sparse, hyphae are transparent, separated, branched, usually wide 2.5-3.7 μ m ; Conidiophores grow from the substrate hyphae, single erect, occasionally branched, 35-200 μm high, 1.5-2.5 μm wide at the base, tapering to the top, 0.5-1.0 μm wide at the top, single conidia Born at the end of the conidiophores; the conidia are transparent, rod-shaped, the distal end is blunt and round, the lower end is tapered, the size is: 22.5-73.8 μm, there are chlamydospores, and the predatory organ is a three-dimensional bacterial net.

The present invention is achieved like this:

Expand the culture of Dactylella shizishanna, YMF1.00022, extract nematicidal active ingredients, and conduct nematicidal efficacy tests to determine its effect on nematodes.

Shizishan Dactylella shizishanna, YMF1.00022 culture method (below is weight percent):

1. Test tube culture

The medium formula is: 1-2% glucose, 10-20% potato juice (obtained by filtering after boiling for 20 minutes), 1.8-2% agar; the pH is natural. Inoculate the mycelium on the culture medium, and cultivate it at 25-28°C for 10-14 days to obtain the test tube species;

2. Liquid expansion culture

Liquid medium formula: 0.1-0.2% gelatin; 0.6-0.8% tryptone; 0.1-0.2% yeast extract powder; 0.05% ammonium sulfate; 0.001-0.002% ferrous sulfate; 0.05-0.1% magnesium sulfate; 1.129% phosphoric acid Disodium hydrogen; 1.238% sodium dihydrogen phosphate pH6.5, the rest is water. Inoculate the test tube seeds into 250ml Erlenmeyer flask liquid culture medium, each bottle contains 60ml, and culture on a shaker at 22-28°C for 6-9 days with a rotation speed of 200-230rpm.

3. Extract nematode active ingredients

Filter the fermentation culture with clean gauze to remove the mycelium, take the supernatant, slowly add ammonium sulfate at a ratio of 1:0.56, let stand at 4°C for 2 hours, then centrifuge at 8500rpm for 15 minutes, and discard the supernatant. Re-dissolve with 10mM phosphate buffer (68.5ml of 10mM sodium dihydrogen phosphate plus 31.5ml of 10mM disodium hydrogen phosphate, pH 6.5), and put the re-dissolved solution into a 21mm dialysis bag (MW: 8,000-14400KDa) in Dialyze in 10-20 times of 10mM phosphate buffer solution for 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 test drugs

Cultivate Dactylella shizishanna, YMF1.00022 strain according to the aforementioned liquid expansion culture method, and prepare test agents according to the aforementioned method of extracting nematicidal active ingredients.

2. Preparation of drugs for comparison

Control 1: Prepare the control agent according to the method for preparing the experimental agent, but the culture medium is not inoculated with Dactylella shizishanna, YMF1.00022 strain.

Control 2: In order to prove that the active ingredient of the nematode is poisonous protein or enzyme, the prepared test agent was boiled and inactivated as a control.

Control 3: clean water was used as the control.

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

4. Test results

Table 1. Insecticidal effect of Alepdactylys spp. YMF1.00022 on Panagrellus redivivus nematodes 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 90 Insect rigidity 98 Body wall mostly dissolved 100 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 twenty one body wall intact

Table 2 Insecticidal effect of Sematympha spp. YMF1.00022 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 40 Insect rigidity 60 Insect rigidity 80 Insect rigidity Control 1 5 body wall intact 10 body wall intact 15 body wall intact Control 2 10 body wall intact 15 body wall intact 25 body wall intact Control 3 11 body wall intact 13 body wall intact twenty two body wall intact

The results show that the test agent extracted from the liquid fermentation product of Dactylella shizishanna, YMF1.00022 strain has a good poisonous effect on Panagrellus redivivus nematodes, and the 24-hour mortality rate of the nematodes reaches 98%. dissolved (Figure 1). 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 agent extracted from the same batch, resulting in protein denaturation, its nematicidal activity is very small, and the nematode body wall does not appear to dissolve (Figure 2), which also proves the activity of S. shizishanii to nematodes The main ingredients are enzymes.

The result shows simultaneously that Dactylella shizishanna, YMF1.00022 bacterial strain has certain poisonous action to pine wood nematode, and the death rate of nematode 36 hours reaches 80%, and worm body is rigid, illustrates that bacterial strain of the present invention is to pine wood nematode The poisonous effect is significantly lower than that of Panagrellus redivivus nematodes, and the action time is delayed.

The Dactylella shizishanna, YMF1.0, 0022 strain of the present invention has significantly higher toxicity to nematodes than other similar microorganisms found so far. At the same time, it grows very quickly 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 their heads intact

Detailed ways:

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

Embodiment one:

The mycelium of Dactylella shizishanna, YMF1.00022 was inoculated on the plate agar medium, and the medium formula was PDA medium formula. Inoculate the mycelium of Dactylella shizishanna, YMF1.00022 on the culture medium, and culture it at 28°C for 10-14 days to obtain the plate species.

Plate seed is inoculated in 250ml Erlenmeyer flask liquid medium (60ml of every bottle), and liquid medium formula is: 0.15% gelatin; 0.8% tryptone; 0.1% yeast extract powder; 0.05% ammonium sulfate; 0.001% ferrous sulfate; 0.05% magnesium sulfate; 1.129% disodium hydrogen phosphate; 1.238% sodium dihydrogen phosphate pH6.5, the rest is water. The culture time was 7 days on a shaker at 27° C., and the rotation speed was 220 rpm.

Filter the fermentation culture with clean gauze to remove the mycelium, take the supernatant, slowly add ammonium sulfate at a ratio of 1:0.56, let stand at 4°C for 2 hours, then centrifuge at 8500rpm for 15 minutes, and discard the supernatant. Re-dissolve with 10mM phosphate buffer (68.5ml of 10mM sodium dihydrogen phosphate plus 31.5ml of 10mM disodium hydrogen phosphate, pH 6.5), and put the re-dissolved solution into a 21mm dialysis bag (MW: 8,000-14400KDa) in Dialyze in 10-20 times of 10mM phosphate buffer solution for 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.

Embodiment two:

It is basically the same as in Example 1, except that the formula of the liquid medium is: 0.1% gelatin; 0.6% tryptone; 0.15% yeast extract powder.

Embodiment three:

It is basically the same as Example 1, except that the formula of the liquid medium is: 0.2% gelatin; 0.7% tryptone; 0.2% yeast extract powder.

Claims (4)

1. A nematode-eating fungus with a nematicidal function, prepared from a production strain through the process of test tube cultivation and liquid fermentation culture, characterized in that the production strain is Dactylella shizishanna (Dactylella shizishanna) YMF1.00022, storage date: 2005 January 27th, deposit number: CGMCC No.1311. 2. The preparation method of the nematode-eating fungus with nematicidal function as claimed in claim 1, comprising in-vitro culture, liquid fermentation culture, obtaining nematode-eating fungus from the liquid fermentation culture, characterized in that the liquid fermentation medium formula used For: 0.1-0.2% gelatin; 0.6-0.8% tryptone; 0.1-0.2% yeast extract powder; 0.05% ammonium sulfate; 0.001% ferrous sulfate; 0.05% magnesium sulfate; 1.129% disodium hydrogen phosphate; Sodium hydrogen has a pH of 6.5, and the remainder is water, and the above are all percentages by weight. 3. The method for extracting nematode active ingredients from the liquid fermentation product of claim 2, comprising: filtering the fermentation culture to remove the mycelium with clean gauze, taking the supernatant, and slowly adding ammonium sulfate in a ratio of 1:0.56 , stand at 4°C for 2 hours, then centrifuge at 8500rpm for 15 minutes, discard the supernatant; redissolve with 10mM phosphate buffer, put the redissolved solution into a 21mm dialysis bag and dialyze in 10-20 times of 10mM phosphate buffer 3-4 times, 3 hours each time. 4. The use of the nematode-eating fungus according to claim 1, characterized in that the fungus has poisonous effect on Panagrellus redivivus nematodes and pine wood nematodes, and can be applied to the biological control of plant parasitic nematodes.