CN104560723B - One plant thick wall bacterium spore Pu Keniya bacterial strains and its screening and application - Google Patents
One plant thick wall bacterium spore Pu Keniya bacterial strains and its screening and application Download PDFInfo
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Abstract
The present invention relates to one plant thick wall bacterium spore Pu Keniya bacterial strains and its screening and application, belong to wireworm-killing biologic resource and microorganism fungus kind technical field.Thick wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) YMF 1.00613 is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on May 10th, 2010, and preserving number is CGMCC No.3806.The screening step of the bacterial strain includes：Strain isolate and purify and strain production aurovertin (aurovertins) class secondary metabolite screening.Application of the thick bacterial strains of wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) YMF 1.00613 of the present invention in the agricultural insecticide and biological pesticide fermenting and producing for preparing preventing and treating plant pathogeny line insect.The effective of the present invention is：Insect breeding and offspring flocks quantity can significantly be suppressed.There is eelworm-killing activity under the ability for killing nematode work, experiment condition, up to more than 90%, can be used in the fermenting and producing of the bioactive substances such as nematicide and biological pesticide for bacterial strain PDB zymotic fluids and its secondary metabolite main component aurovertin class compound.
Description
Technical field：
The present invention relates to one plant thick wall bacterium spore Pu Keniya bacterial strains and its screening and application, belong to wireworm-killing biologic resource and
Microorganism fungus kind technical field.
Background technology：
Plant pathogeny line insect is a kind of to endanger serious plant disease, it was reported that plant pathogeny line insect belong to up to more than 200
More than 5000 plant.Plant pathogeny line insect disease brings nearly 157,000,000,000 dollars of economic loss to whole world agricultural every year, is only second to true
Fungus diseases.At present, chemical prevention is still to control one of important measures of Plant nematode, but the hypertoxic chemistry of long-term use kills nematode
Agent increasingly appears to the harm that environment and the health care belt of the mankind come.Therefore, the GR of high-efficiency low-toxicity has been researched and developed
Major issue as agricultural sustainable development.
Biocontrol of nematodes is attracted attention, and national governments also give very big support, and first generation nematode is biological
The live bacteria agent of control agent such as nematode-trapping fungi, inner parasitic epiphyte and parasitics bacterium arise at the historic moment, but it is by environmental factor shadow
The problems such as ringing big, less stable, short shelf life, is restricted its application.To overcome this disadvantage, domestic and international researcher is more
Second generation nematode bio-control factors i.e. Metabolite is paid attention in research and develop.The secondary metabolite of fungi is used as second generation line
Worm bio-control factors, are played a role in the way of directly acting on crops nematode, there is fast effect, drug effect height, Environmental compatibility
Well, the advantages of cannot be easily caused secondary pollution.In recent years because nematode, fungi etc. cause the various cause of diseases of plant infectious diseases
The resistance of thing, original agricultural antibiotic, nematode killing agent antibiotic therapy effect in decline, it is necessary to continually develop new antibiotic,
And it is the key of problem to find new natural antibiotics active ingredient.
The content of the invention：
It is an object of the present invention to overcome not enough and nematicide present in existing nematicide efficiently to produce bacterium
The problem of planting scarcity of resources, filters out from the fungi with poisoning plant pathogeny line insect function with generation eelworm-killing activity
The thick wall bacterium spore Pu Keniya bacterium (Pochonia of the bacterial strain of aurovertin (aurovertins) class secondary metabolite
Chlamydosporia) YMF 1.00613, are that research and development biological mematocide and the bionical biological pesticide of research and development are established
Basis.
Second mesh of the invention is to provide thick wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia)
The screening technique of YMF1.00613 bacterial strains.
3rd mesh of the invention is to provide thick wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia)
The application of YMF1.00613 bacterial strains.
To achieve the above object, the invention provides following technical scheme:
Thick wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) YMF 1.00613 of the present invention is in 2010
On May 10, in is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address：Beijing
The institute 3 of Chaoyang District Bei Chen West Roads 1, Institute of Microorganism, Academia Sinica, preserving number is CGMCC No.3806.Thick wall bacterium spore
Pu Keniya bacterium (Pochonia chlamydosporia) YMF 1.00613 belongs to Pu Keni subgenus (Pochonia spp.),
It is once called as Verticillium or Verticillium dahliae category (Verticillium spp.).The category fungi being capable of parasitic root-knot nematode
(Meloidogyne spp.), pseudo- knot nematodes (Nacobbus spp.) and SCN (Heterodera spp.,
Globodera spp.) ovum and female adult, can significantly suppress insect breeding and offspring flocks quantity.
The thick bacterial strains of wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) YMF 1.00613 of the present invention
Screen step as follows：
(1) strain is isolated and purified
The root-knot nematode gathered with building shredded tobacco for water pipes from Yunnan is infected into tobacco old complaint ± 300g root knots and is cut into 3-5cm trifles, is put
Test tube is received below the graceful funnel device of Bel for filling water, funnel with a rubber tube and nematode to connect, nematode is in Action of Gravity Field
Ttom of pipe is sunken to, after 24 hours, rubber tube is clamped, reception pipe is removed, 8000rpm centrifugations abandon supernatant, obtain nematode suspension 1mL；Take
0.2mL nematode suspensions are coated on 2% agar level board, and 28 DEG C are cultivated 10~20 days；When there is bacterium colony formation, chosen from colony edge
Take a little mycelia to move to new PDA plate, cultivate ± 7 days, observe the colonial morphology grown up to, carry out Preliminary Identification, and by bacterium colony
It is transferred on CMA flat boards and purifies culture, CMA culture mediums is：Corn flour 30g boils 30min, filters to get filtrate, and adds agar
15g, is added water and is settled to 1L, and heating is mixed, and 121 DEG C sterilize 20 minutes after packing；
(2) screening of production aurovertin (aurovertins) class secondary metabolite of strain
By potato 200g, glucose 20g, agar 15g, changed into after water 1000mL, natural PH PDA culture medium sterilizing flat
Plate, by 1cm2The fungus blocks of fungi YMF 1.00613 are inoculated on flat board, and 28 DEG C are cultivated 7 days；Flat board strain is inoculated into per bottled
In the 500mL triangular flasks of 250mL PDB fluid nutrient mediums, PDB fluid nutrient mediums are：Potato 200g, glucose 20g, water
1000mL, nature PH, the shaking table culture at 28 DEG C, rotating speed 180rpm, incubation time 12 days；Using filter paper by bacterium solution and mycelium
Separation, filtrate is after fermenation raw liquid is concentrated under reduced pressure, to be extracted three times with isometric ethyl acetate, is then combined with ethyl acetate decompression steaming
It is dry；Mycelium ethanol or acetone soak are after 12 hours, by methanol extract liquid evaporated in vacuo；Hair is soaked respectively with 1mL acetone
Zymotic fluid ethyl acetate portion and mycelium methanol crude extract, ultrasonic wave 20min carry out the detection of thin-layer chromatography TLC plates, with chloroform：First
Alcohol=15:1 be panel system when, UV detect when show yellow-green fluorescence under 365nm, blackening, 10% are shown under 254nm
H2SO4It is purple when alcoholic solution sprays and heats colour developing, several similar bands (See Figure 1) is detected, wherein in mycelium
The content highest of (M in Fig. 1) compound 4 in methanolic extract, in the content of zymotic fluid ethyl acetate portion (C in Fig. 1) compound 3
Highest.Four kinds of Aurovertin metabolins compound 1-4 structure is as shown in Figure 2.
The present invention's kills the thick wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) of nematode filamentous fungi
Application of the YMF1.00613 bacterial strains in the agricultural insecticide and biological pesticide fermenting and producing for preparing preventing and treating plant pathogeny line insect.
The effective of the present invention is：Insect breeding and offspring flocks quantity can significantly be suppressed.Bacterial strain PDB zymotic fluids
And its secondary metabolite main component aurovertin class compound has the ability for killing nematode work, nematode work is killed under experiment condition
Property can be used in the fermenting and producing of the bioactive substances such as nematicide and biological pesticide up to more than 90%.
Brief description of the drawings：
Fig. 1 shows TLC detections P.chlamydosporia zymotic fluid ethyl acetate portion (C) and mycelium methanol crude extract
(M) result.
Fig. 2 is the structure chart of compound 1-4.
Fig. 3 shows the knot of the zymotic fluid aurovertin class contents of HPLC detection P.chlamydosporia YMF 1.00613
Really.
Embodiment：
Thick wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) YMF 1.00613 that the present embodiment is used
China Committee for Culture Collection of Microorganisms's common micro-organisms center is deposited on May 10th, 2010, preserving number is
CGMCC-3806。
The thick wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) of nematicidal fungi that the present embodiment is used
The screening step of YMF1.00613 bacterial strains is as follows：
(1) strain is isolated and purified
Root-knot nematode is gathered from Yunnan with building shredded tobacco for water pipes and infects tobacco old complaint sample, uses plastic bag sealing.General ± 300g root knots
3cm or 5cm trifles are cut into, is put in below the graceful funnel device of Bel for filling water, funnel and receives test tube with a rubber tube and nematode
Connect, nematode then can be sunken to ttom of pipe due to Action of Gravity Field.After 24 hours, rubber tube is clamped, reception pipe, 8000rpm centrifugations is removed
Supernatant is abandoned, nematode suspension 1mL is obtained.0.2mL nematodes suspension (containing ± 200 active or standing nematodes) is taken to be coated on 2% agar water
Flat board, 28 DEG C are cultivated 10 days or 15 days or 20 days.When there is bacterium colony formation, moved to from a little mycelia of colony edge picking new
PDA plate, is cultivated ± 7 days, observes the colonial morphology grown up to, carries out Preliminary Identification, and bacterium colony is transferred into (CMA on CMA flat boards
Culture medium is：Corn flour 30g boils 30min, filters to get filtrate, and adds agar 15g, adds water and be settled to 1L, and heating is mixed, packing
121 DEG C sterilize 20 minutes afterwards) purifying culture.
Colony characteristicses：Purify obtained bacterium colony and grow comparatively fast that (PDA culture medium is in PDA culture medium:Potato 200g,
Glucose 20g, agar 15g, water 1000mL, natural PH, flat board is changed into after sterilizing), the colony diameter of culture 10 days is 8cm, in vain
Color villiform, the shallow yellow to brown in the back side；Conidiophore is verticillate, bottle stalk jumping chisel type.Conidium clear, colorless, it is near avette, ellipse
Circle, unicellular, size is 3-4.5 × 1.5-2.2 μm, and chlamydospore is light yellow, muriform, many cells, 20-25 μm of diameter.
(2) screening of production aurovertin (aurovertins) class secondary metabolite of strain
By potato 200g, glucose 20g, agar 15g, changed into after water 1000mL, natural PH PDA culture medium sterilizing flat
Plate, by 1cm2The fungus blocks of fungi YMF 1.00613 are inoculated on flat board, and 28 DEG C are cultivated 7 days；Flat board strain is inoculated into per bottled
In the 500mL triangular flasks of 250mL PDB fluid nutrient mediums, PDB fluid nutrient mediums are：Potato 200g, glucose 20g, water
1000mL, nature PH, the shaking table culture at 28 DEG C, rotating speed 180rpm, incubation time 12 days；Using filter paper by bacterium solution and mycelium
Separation, filtrate is after fermenation raw liquid is concentrated under reduced pressure, to be extracted three times with isometric ethyl acetate, is then combined with ethyl acetate decompression steaming
It is dry；Mycelium ethanol or acetone soak are after 12 hours, by methanol extract liquid evaporated in vacuo；Hair is soaked respectively with 1mL acetone
Zymotic fluid ethyl acetate portion and mycelium methanol crude extract, ultrasonic wave 20min carry out the detection of thin-layer chromatography TLC plates, with chloroform：First
Alcohol=15:1 be panel system when, UV detect when show yellow-green fluorescence under 365nm, blackening, 10% are shown under 254nm
H2SO4It is purple when alcoholic solution sprays and heats colour developing, several similar bands (see Fig. 1) is detected, wherein in mycelium first
The content highest of (M in Fig. 1) compound 4 in alcohol extracting thing, in the content of zymotic fluid ethyl acetate portion (C in Fig. 1) compound 3 most
It is high.Four kinds of Aurovertin metabolins compound 1-4 structure is as shown in Figure 2.
The application of the bacterial strain fermentation liquor of the present invention is as follows：
Evaluated by the nematode ability of killing to bacterial strain, the nematode is plant root-knot nematode and saprophitic nematode.South
Root-knot nematode Meloidogyne incognita (race 3) cultures (± 25 DEG C) tomato root in greenhouse.Collection carries root
The tomato diseased plant of tie lines worm's ovum block, ± 2cm segments are cut into by old complaint, with the ripe pieces of an egg of the dissecting needle picking of sterilizing and are put
In 40 μm of cell screen clothes.Screen cloth and pieces of an egg are placed in 1min in 2% liquor natrii hypochloritis, during which weak vibrations screen cloth.Sterilized water
Rinse that screen cloth is positioned in the 6cm culture dishes equipped with 15mL sterilized waters in 28 DEG C of constant incubators after pieces of an egg 5 times or 7 times and hatch
Obtaining second instar larvae (J2) within 24 hours is used for toxotest.Saprophitic nematode Caenorhabditis elegans Caenorhabditis
Elegans is incubated at NGM culture mediums (the NaCl 3g, peptone 2.5g, cholesterol 5mg/mL for being inoculated with E.coli OP50
1mL, water 975mL, MgSO is added after sterilizing41M 1mL,CaCl21M1mL, phosphate buffer 25mL) on, by culture of nematodes extremely
Obtaining synchronized L1 larvas using alkaline lysis after adult is used for toxotest.
Changed into after PDA culture medium (potato 200g, glucose 20g, agar 15g, water 1000mL, natural PH) is sterilized flat
Plate.Fungi fungus block is inoculated on flat board, 28 DEG C are cultivated 7 days.Flat board strain is inoculated into 500mL triangular flasks (per bottled
250mL) PDB fluid nutrient mediums (potato 200g, glucose 20g, water 1000mL, natural PH), the shaking table culture at 28 DEG C turns
Fast 180rpm, incubation time 12 days.Bacterium solution is separated with mycelium using filter paper, bacterium solution is sterilized 120 DEG C, 20min.
With the eelworm-killing activity of liquid immersion method bacterium zymotic fluid：Using 24 porocyte culture plates, in every Kong Zhongjia
Enter 1mL ferment filtrates and 10 μ L nematodes suspensions (average 500 nematodes/10 μ L), 20 DEG C (C.elegans) or 28 DEG C
(M.incognita) after standing 24 hours, nemic death rate is counted under the microscope.Identify that the dead method of nematode is, it is dead
Nematode is stiff or in " J " type, and living nematode then crimps twisting.Each portion of static nematode body is stirred with needle point, nematode is not taken the post as yet
What reaction is then accredited as death.
The death rate=(verge of death borer population/(verge of death borer population+hot line borer population)) × 100%
Corrected mortality=test sample the death rate-control group death rate
Using the PDB fluid nutrient mediums of non-inoculated fungi as control, whole experiment in triplicate, takes three average values, calculates
Go out average mortality and corrected mortality.
As shown in table 1, PDB fermentation liquor treatment plant nematode M.incognita of the bacterial strains of YMF 1.00613 and saprophytic
After nematode C.elegans 24 hours, the death rate of nematode is above 90%, illustrates that the zymotic fluid of this plant of bacterium shows very strong poison
Eelworm-killing activity.Therefore this research screening obtains and kills the thick wall bacterium spore Pu Keniya bacterium of nematode filamentous fungi with kill nematode function
The bacterial strains of YMF 1.00613.
The 24 hours Toxic test results of strains tested fermentation liquor treatment nematode of table 1
(4) application for killing nematode ability of bacterial strain crude extract, to contain aurovertin (aurovertins) class secondary metabolism
Product kills nematode ability to bacterial strain crude extract for index and is estimated, specific as follows：
Thick wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) belong to Pu Keni subgenus (Pochonia
Spp.), it is once called as Verticillium or Verticillium dahliae category (Verticillium spp.).There is the category fungal bacterial strain generation to kill nematode etc.
The performance of active material.Therefore it can apply to kill the fermenting and producing such as nematode biological prevention and control agent, biological pesticide and antibiotic field.
The seed culture of fungal bacterial strain and the liquid fermentation and culture of fungal bacterial strain：Ibid.By filtering fermentation liquor, filtrate is hair
After ferment stoste is concentrated under reduced pressure, with ethyl acetate equal-volume extraction three times, evaporated in vacuo after then extract is merged.By crude extract
Silica gel chromatograph post separation is carried out, with methanol/chloroform (9:1) be eluent, collect eluent, be concentrated under reduced pressure into it is dry, then
Freeze-drying obtains yellow powder crude extract in 3 hours.
Aurovertin in different incubation time fungal mycelium fermentation broth coarse extracts is detected using HPLC-DAD
(aurovertins) content of each monomeric compound of class secondary metabolite, (according to aurovertinDEIF concrete numerical value meters
The ratio that aurovertinD accounts for aurovertinD+E+I+F is calculated, is as a result shown after 10 days shared by compound D content
Aurovertins ratio highests, are shown in Fig. 3.
The eelworm-killing activity of bacterial strain aurovertin class secondary metabolite crude extract is detected with liquid immersion method, gold wheel is mould
Plain class secondary metabolite crude extract is dissolved in DMSO, and 400 μ g/mL, 200 μ g/mL, 100 μ g/mL, 50 μ g/mL and 25 are prepared respectively
The test liquid of μ g/mL concentration, DMSO final concentration is no more than 0.5% in the solution.Separately the DMSO of same concentrations is dissolved in
The aqueous solution is used as control.The cultural method of Meloidogyne incognita M.incognita (race 3) second instar larvae (J2) is ibid.By corruption
Raw nematode Panagrellus redivivus P.redivevus is inoculated on oat medium (oat 20g, water 80mL), is cultivated 5 days in 28 DEG C
Or 7 days.Nematode needed for being washed out with modified Baermann funnel method, it is with sterile distilled water that nematode is dilute after 1% hypochlorite disinfectant 2min
6000/mL is interpreted as nematode suspension.Test solution 1mL is put in 3.5cm flat boards, living nematode 1500 is added, is placed in
28 DEG C of constant incubators, observation line polypide wall is cleared up under the microscope situation simultaneously counts nemic death rate.Identify that nematode is dead
Method and calculate average mortality and corrected mortality ibid.Experimental result is as follows：
Aurovertin class secondary metabolite crude extract is the main component in the bacterial strain fermentation liquors of YMF 1.00613, toxicity
Test result (being shown in Table 2-3) shows aurovertin class secondary metabolite crude extract to root-knot nematode M.incognita and saprophytic
Nematode P.redivevus LC50 (semilethal rate) was respectively 48.5 μ g/mL at 24 hours-1With 50.6 μ g/mL-1, have to nematode
There is good cytotoxicity.
The test sample of table 2 handles M.incognita 24 hours Toxic test results of J2
The test sample of table 3 handles P.redivevus Toxic test results
Claims (2)
1. the thickness bacterial strains of wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) YMF 1.00613, the preservation of the bacterial strain
Number it is：CGMCC No.3806.
2. the thick bacterium of wall bacterium spore Pu Keniya bacterium (Pochonia chlamydosporia) YMF 1.00613 described in claim 1
Application of the strain in the agricultural insecticide and biological pesticide fermenting and producing for preparing preventing and treating plant pathogeny line insect.
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| CN106148447B (en) * | 2016-07-01 | 2018-10-23 | 杭州唯铂莱生物科技有限公司 | A kind of bioconversion synthetic method of ortho position chloro hydroxy benzo pyridine |
| CN106566776B (en) * | 2016-10-10 | 2019-09-24 | 中国林业科学研究院森林生态环境与保护研究所 | Chlamydospores Pukeniya bacteria Flight Mutagenesis mutant strain Pc-m-6 and its biological prevention and control agent and application |
| CN107022495B (en) * | 2017-04-21 | 2020-04-14 | 云南大学 | Two strains of Pochonia chlamydosporia fungus and application thereof |
| CN107955802A (en) * | 2017-12-05 | 2018-04-24 | 福建农林大学 | A kind of method of thickness wall spore Pu Keniya bacteria solid fermentations production spore |
| CN116875499B (en) * | 2023-07-06 | 2024-04-09 | 吉林省农业科学院 | Entomopathogenic nematode symbiotic bacteria and application of metabolite thereof |
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| CN102031277A (en) * | 2009-12-09 | 2011-04-27 | 云南大学 | Aurovertin metabolins and application thereof |
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| CN102031277A (en) * | 2009-12-09 | 2011-04-27 | 云南大学 | Aurovertin metabolins and application thereof |
| CN102321538A (en) * | 2011-07-12 | 2012-01-18 | 云南大学 | Microbial composite inoculum and applications thereof |
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| 厚垣轮枝孢菌(Verticillium chlamydosporium;卢明科等;《西北农林科技大学学报（自然科学版）》;20040430;第32卷(第4期);全文 * |
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