CN1288239C - Nematode-eating fungus with nematode-killing function, preparing method and use thereof - Google Patents
Nematode-eating fungus with nematode-killing function, preparing method and use thereof Download PDFInfo
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- CN1288239C CN1288239C CN 200510010689 CN200510010689A CN1288239C CN 1288239 C CN1288239 C CN 1288239C CN 200510010689 CN200510010689 CN 200510010689 CN 200510010689 A CN200510010689 A CN 200510010689A CN 1288239 C CN1288239 C CN 1288239C
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Abstract
The present invention relates to a nematode-eating fungus with a nematode filling function and a preparation method and an application thereof, which belongs to the technical field of microbial pesticides. Effective ingredients for filling nematodes are extracted from metabolic products after a strain is fermented in liquid, and the effective ingredients are prepared into the nematode-eating fungus. A production strain is dactylella shizishanna)YMF1.00022. The formula of a liquid fermentation culturing medium of the production strain comprises the ingredients of 0.1 to 0.2 % of gelatin, 0.6 to 0.8% of tryptone, 0.1 to 0.2% of yeast extract powder, 0.05% of ammonium sulfate, 0.001% of ferrous sulfate heptahydrate, 0.05% of magnesium sulfate, 1.129% of disodium hydrogenorthophosphate, 1.238% of sodium dihydrogenorthophosphate (pH6.5) and water as the rest. The method of extracting the effective ingredients for killing nematodes comprises: supernatant fluid is taken from a fermentation culturing product, ammonium sulfate is slowly added to the supernatant fluid according to the proportion of 1 to 0.56, and the supernatant fluid is statically placed for two hours at the temperature of 4 DEG C; next, the supernatant fluid is centrifugated for 15 minutes at 8500 rpm, and the supernatant state is eliminated. The supernatant fluid is dissolved again in phosphate buffer fluid of 10mM, and an obtained solution formed by resolution is filled in a dialysis bag of 21mm and is dialyzed for 3 to 4 times in 10 to 20 times of the phosphate buffer fluid of 10mM, wherein the solution is dialyzed for 3 hours in each time. The nematode-eating fungus has the obvious advantage of high toxicity to nematodes, and has good potential application prospects.
Description
Technical field:
The present invention relates to a kind of Nematophagous fungi and its production and application, the microorganism belonging to genus technical field of pesticide with nematode killing function.
Background technology:
Plant nematode is the Plant diseases that generally takes place in a kind of world wide, and only the known kind of root knot nematode reaches kind more than 70,3000 various plants that cause harm, and the loss that causes every year is huge.According to Food and Argriculture OrganizationFAO's statistics, cause damage the whole world up to 1,000 hundred million dollar because of nematode every year.Main cash crop such as China nematode main harm tobacco, flowers, vegetables, cotton, soybean, peanut, pseudo-ginseng, Radix Panacis Quinquefolii become the critical limitation factor that develops these crops.For a long time, control of nematode mainly depends on chemical prevention.Although chemical nematocides has been brought into play vital role in control of nematode,, find that many chemical nematocidess all have side effect along with progress of science and technology.Cause environmental pollution, be detrimental to health, have many disabled or be about to forbidding in succession.Particularly plant nematode betides crop root, because the singularity of edatope, must heavy dose uses and could guarantee its preventive effect.And heavy dose of chemical pesticide is very serious to groundwater pollution.Therefore, research and development high-efficiency low-toxicity low residue nematocides has been the important topic in the agricultural sustainable development.
Food nematode fungus is a class nemic natural enemy, the nematode biological prevention and control agent that success is in the market gone on the market this class fungus of all originating.Wherein, every referring to that the spore bacterium is important monoid.
Summary of the invention:
The objective of the invention is to decompose of the research of the Lion Rock of nematode body wall, develop biological nematocides every finger spore fungi by a strain being produced extracellular protease.
Lion Rock is every referring to that spore is in the food nematode fungus research carried out of the inventor, a strain of from a large amount of samples, separate finding to nematode have fine toxic action every referring to that spore belongs to novel species, the called after Lion Rock is every referring to spore (Dactylellashizishanna) YMF1.00022, and the YMF1.00022 bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center; Address: China. Beijing. the Zhong Guan-cun; Preservation date: on January 27th, 2005; The numbering CGMCC No.1311 that preservation is registered on the books.
Lion Rock of the present invention is every referring to that spore (Dactylella shizishanna) YMF1.00022 strain morphology is characterized as: bacterium colony is closely colourless on the CMA substratum, and aerial hyphae is sparse, and mycelia is transparent, separates branch, common wide 2.5-3.7 μ m; Conidiophore grows from the matrix mycelia, and single upright, idol has branch, high 35-200 μ m, and the wide 1.5-2.5 μ of base portion m, gradually thin to the top, top wide 0.5-1.0 μ m, single conidium and is born in the conidiophore end; Conidium is transparent, and is bar-shaped, the distal blunt circle, and the lower end is gradually thin, and size is: 22.5-73.8 μ m, chlamydospore is arranged, trapping organs is three-dimensional bacterium net.
The present invention is achieved in that
The enlarged culturing Lion Rock is every referring to spore Dactylella shizishanna, and YMF1.00022 extracts nematicide effective constituent, carries out the test of nematicide drug effect, determines its effect to nematode.
Lion Rock is every referring to spore Dactylella shizishanna, YMF1.00022 cultural method (below be weight percentage):
1, the test tube kind is cultivated
Culture medium prescription is: 1-2% glucose, 10-20% potato juice (boiling 20 minutes after-filtration gained), 1.8-2% agar; The pH nature.Mycelium is inoculated on the substratum, cultivated 10-14 days down, obtain the test tube kind for 25-28 ℃;
2, fluid enlargement culture
The liquid culture based formulas is: the 0.1-0.2% gelatin; The 0.6-0.8% Tryptones; The 0.1-0.2% yeast extract powder; 0.05% ammonium sulfate; The 0.001-0.002% ferrous sulfate; 0.05-0.1% sal epsom; 1.129% Sodium phosphate dibasic; 1.238% SODIUM PHOSPHATE, MONOBASIC pH6.5, remainder is a water.The test tube kind is inoculated in the 250ml triangular flask liquid nutrient medium, and every bottled 60ml cultivated incubation time 6-9 days in 22-28 ℃ of following shaking table, and rotating speed is 200-230rpm.
3, extract the eelworm-killing activity composition
Fermenting culture with clean gauze elimination mycelium, is got supernatant liquor, slowly add ammonium sulfate in 1: 0.56 ratio, left standstill 2 hours in 4 ℃, 8500rpm is centrifugal 15 minutes then, abandons supernatant.(the 10mM SODIUM PHOSPHATE, MONOBASIC of 68.5ml adds the 10mM Sodium phosphate dibasic of 31.5ml with the 10mM phosphoric acid buffer, pH is 6.5) dissolving again, to dissolve the solution that the obtains 21mm dialysis tubing (MW:8 that packs into again, 000-14400KDa) in 10-20 10mM phosphoric acid buffer doubly, dialyse 3-4 time, each 3 hours, promptly get the nematicide soup.Be stored in 4 ℃ of refrigerators soup stand-by.
The eelworm-killing activity test:
1, preparation test with medicament
Cultivate Lion Rock every referring to spore Dactylella shizishanna by aforementioned liquids enlarged culturing method, the YMF1.00022 bacterial strain is by the method preparation test with medicament of aforementioned extraction eelworm-killing activity composition.
2, preparation contrast with medicament
Contrast 1:, but do not insert Lion Rock in the substratum every referring to spore Dactylella shizishanna, YMF1.00022 bacterial strain by aforementioned preparation test with medicament method preparation contrast with medicament.
Contrast 2: in order to prove the nematicide effective ingredient is toxalbumin or enzyme, will prepare reagent agent and boil after the deactivation in contrast.
Contrast 3: in contrast with clear water.
3, nematode is used in the preparation test
1) preparation Panagrellus redivivus nematode
The P.redivivus nematode is inoculated on the medium oatmeal, cultivated 6 days down, freeze in 4 ℃ of refrigerators standby in 28 ℃.Required nematode is washed out with the graceful funnel method of shellfish, place in the 5ml centrifuge tube, add the washing of 5ml sterilized water, instantaneous centrifugal, abandon supernatant, repeat 5 times and obtain clean for the examination nematode.Is that content is the nematode suspension of 15/μ l with sterilized water with the nematode dilution.
2) preparation pine wood nematode (Bursaphelenchus xylophilus)
Put into 15g through 2 days corn grain of water logging bubble in the 100ml triangular flask, add water 10ml, autoclaving inserts Botrytis cinerea (Botrytis cinerea).Cultivated 4 to 7 days for 25 ℃.After treating that mycelia is paved with triangular flask, inoculation was cultivated 15 to 20 days for 28 ℃ through the pine wood nematode of 0.25% clorox surface sterilization.With sterilized water nematode is washed, making content is the nematode suspension of 15/μ l.
3, test method:
(1) test of pesticide effectiveness method
Get test with medicament 200 μ l in the 1.5ml centrifuge tube, add 60 of Panagrellus redivivus nematode and pine wood nematodes respectively, centrifuge tube keeps flat, and places under 25 ℃, respectively at the mortality ratio of checking the calculating nematode in 12 hours, 24 hours, 36 hours.And under opticmicroscope, observe observation line polypide wall changing conditions
Identify that dead method is: add 1-5 and drip 5%Nacl solution in handling centrifuge tube, observe after 2 minutes, dead worm is stiff, and the worm that lives is then curled or twisting.
Respectively with 3 kinds of contrast medicaments, every processing triplicate.
4, test-results
Table 1 Lion Rock is every referring to that spore YMF1.00022 is to Panagrellus redivivus nematode insecticidal effect
| Chemicals treatment | The nematode death condition that is fixed | |||||
| 12 hours | 24 hours | 36 hours | ||||
| Mortality ratio % | Nematode changes | Mortality ratio % | Nematode changes | Mortality ratio % | Nematode changes | |
| The test with medicament | 90 | Polypide is stiff | 98 | Body wall dissolves mostly | 100 | Exhausted most dissolving |
| Contrast 1 | 10 | No change | 20 | Not dissolving | 30 | Body wall is complete |
| Contrast 2 | 10 | No change | 10 | Not dissolving | 20 | Body wall is complete |
| Contrast 3 | 10 | No change | 10 | Not dissolving | 21 | Body wall is complete |
Table 2 Lion Rock is every referring to that spore YMF1.00022 is to the pine wood nematode insecticidal effect
| Chemicals treatment | Nematode is fixed and death condition | |||||
| 12 hours | 24 hours | 36 hours | ||||
| Mortality ratio (%) | Nematode changes | Mortality ratio (%) | Nematode changes | Mortality ratio (%) | Nematode changes | |
| The test with medicament | 40 | Polypide is stiff | 60 | Polypide is stiff | 80 | Polypide is stiff |
| Contrast 1 | 5 | Body wall is complete | 10 | Body wall is complete | 15 | Body wall is complete |
| Contrast 2 | 10 | Body wall is complete | 15 | Body wall is complete | 25 | Body wall is complete |
| Contrast 3 | 11 | Body wall is complete | 13 | Body wall is complete | 22 | Body wall is complete |
The result shows, from Lion Rock every referring to spore Dactylella shizishanna, the test with medicament of extracting in the YMF1.00022 strain liquid tunning is to Panagrellus redivivus nematode tool toxic action preferably, and 24 hours mortality ratio of nematode reaches 98%, and body wall mostly dissolves (Fig. 1).From handling the variation of nematode, experienced from the part body wall being dissolved into the whole dissolved processes of polypide, illustrate that active ingredient is mainly the enzyme that decomposes nematode.And will be with batch test with medicament of extracting through boiling, cause protein denaturation after, its eelworm-killing activity is very little, and dissolution phenomena (Fig. 2) do not appear in the nematode body wall, this has proved that also Lion Rock is every referring to that the active ingredient of spore to nematode is mainly enzyme.
The result shows simultaneously, Lion Rock is every referring to spore Dactylella shizishanna, the YMF1.00022 bacterial strain has certain toxic action to pine wood nematode, 36 hours mortality ratio of nematode reaches 80%, polypide is stiff, illustrate that bacterial strain of the present invention is starkly lower than Panagrellus redivivus nematode to the toxic action of pine wood nematode, delay action time.
Lion Rock of the present invention is every referring to spore Dactylella shizishanna, YMF1.0, and 0022 bacterial strain is significantly higher than other same quasi-microorganisms of finding at present to the virulence of nematode.Growth is very fast under culture condition of the present invention simultaneously, has possessed good application and development prospect.
Description of drawings:
Fig. 1 display process nematode is decomposed situation.
Fig. 2 shows the contrast nematode that the contrast head remains intact
Embodiment:
Below be embodiments of the invention, but content of the present invention is not limited thereto.
Embodiment one:
Every referring to spore Dactylella shizishanna, the YMF1.00022 mycelium is inoculated on the dull and stereotyped nutrient agar with Lion Rock, and culture medium prescription is the PDA culture medium prescription.Every referring to spore Dactylella shizishanna, the YMF1.00022 mycelium is inoculated on the substratum with Lion Rock, cultivates 10-14 days down, obtains dull and stereotyped the kind for 28 ℃.
The flat board kind is inoculated into (every bottled 60ml) in the 250ml triangular flask liquid nutrient medium, and the liquid culture based formulas is: 0.15% gelatin; 0.8% Tryptones; 0.1% yeast extract powder; 0.05% ammonium sulfate; 0.001% ferrous sulfate; 0.05% sal epsom; 1.129% Sodium phosphate dibasic; 1.238% SODIUM PHOSPHATE, MONOBASIC pH6.5, remainder is a water.Cultivated incubation time 7 days in 27 ℃ of following shaking tables, rotating speed is 220rpm.
Fermenting culture with clean gauze elimination mycelium, is got supernatant liquor, slowly add ammonium sulfate in 1: 0.56 ratio, left standstill 2 hours in 4 ℃, 8500rpm is centrifugal 15 minutes then, abandons supernatant.(the 10mM SODIUM PHOSPHATE, MONOBASIC of 68.5ml adds the 10mM Sodium phosphate dibasic of 31.5ml with the 10mM phosphoric acid buffer, pH is 6.5) dissolving again, to dissolve the solution that the obtains 21mm dialysis tubing (MW:8 that packs into again, 000-14400KDa) in 10-20 10mM phosphoric acid buffer doubly, dialyse 3-4 time, each 3 hours, promptly get the nematicide soup.Be stored in 4 ℃ of refrigerators soup stand-by.
Embodiment two:
Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is: 0.1% gelatin; 0.6% Tryptones; 0.15% yeast extract powder.
Embodiment three:
Substantially with embodiment one, difference is the liquid culture based formulas, and its prescription is: 0.2% gelatin; 0.7% Tryptones; 0.2% yeast extract powder.
Claims (4)
1, a kind of Nematophagous fungi with nematode killing function, by producing bacterial strain through the cultivation of test tube kind, the preparation of liquid fermentation and culture operation, it is characterized in that producing bacterial strain is that Lion Rock is every referring to spore (Dactylella shizishanna) YMF1.00022, preservation date: on January 27th, 2005, preserving number: CGMCC No.1311.
2, the described preparation method of claim 1 with Nematophagous fungi of nematode killing function, comprise the cultivation of test tube kind, liquid fermentation and culture, from the liquid fermentation and culture thing, obtain Nematophagous fungi, it is characterized in that used liquid fermentation medium prescription is: the 0.1-0.2% gelatin; The 0.6-0.8% Tryptones; The 0.1-0.2% yeast extract powder; 0.05% ammonium sulfate; 0.001% ferrous sulfate; 0.05% sal epsom; 1.129% Sodium phosphate dibasic; 1.238% SODIUM PHOSPHATE, MONOBASIC pH6.5, remainder is a water, more than all are weight percents.
, Accessory Right requires to extract in 2 the liquid fermentation production method of nematicide effective constituent, comprising: fermenting culture with clean gauze elimination mycelium, is got supernatant liquor, slowly add ammonium sulfate in 1: 0.56 ratio, left standstill 2 hours in 4 ℃, 8500rpm is centrifugal 15 minutes then, abandons supernatant; Again dissolve with the 10mM phosphoric acid buffer, the 21mm dialysis tubing of packing into of the dissolving solution that obtains is again dialysed each 3 hours 3-4 time in 10-20 10mM phosphoric acid buffer doubly.
4, the purposes of the described Nematophagous fungi of claim 1 is characterized in that this fungi 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100436575C (en) * | 2006-04-26 | 2008-11-26 | 云南大学 | Heteromorphic Dactlella bacterial agent capable of killing nematode and application thereof |
| CN1891812B (en) * | 2006-05-25 | 2010-09-08 | 沈阳农业大学 | Aspergillus fungus with nematode-killing vitality, and its preparing method and use |
| CN101831388B (en) * | 2010-05-18 | 2012-08-22 | 华南农业大学 | Nematophagous fungus and preparation method and application thereof |
| CN102604838B (en) * | 2011-12-23 | 2013-07-10 | 内蒙古大学 | Arthrobotrys oligospora strain N and application thereof |
| CN104450538B (en) * | 2014-11-26 | 2017-07-07 | 中国农业科学院生物技术研究所 | Mould culture medium and cultural method in a kind of Jue Shi plums for circular cone |
| CN104928192A (en) * | 2015-06-26 | 2015-09-23 | 云南大学 | Trichoderma viride strain and application thereof |
| CN109197900B (en) * | 2017-07-04 | 2021-10-29 | 北京国康本草物种生物科学技术研究院有限公司 | Compound biological agent and application thereof in eliminating root-knot nematodes |
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