CN102925369A - Chaetomium globosum with nematicidal activity, metabolite and application thereof - Google Patents
Chaetomium globosum with nematicidal activity, metabolite and application thereof Download PDFInfo
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Abstract
The invention relates to a chaetomium globosum with nematicidal activity, a metabolite and an application of the chaetomium globosum, belonging to the field of microbial pesticide technology. The preservation number of the chaetomium globosum NK106 is CGMCC6716. The invention includes the application of the chaetoglobosinA(ChA) generated by the bacterial strain in preventing the plant nematode. ChA has outstanding characteristics of high toxicity and remarkable nematicidal effect for Meloidogyne incognita. According to the invention, the effect of the strain fermentation broth and purified ChA on second-stage juveniles (J2) of Meloidogyne incognita is detected. The treatments of fermentation in different dilution concentrations have significant differences from the control treatment. The fermentation liquid and ChA has a lethal effect on the J2. When the concentration of the ChA is 300mug/mL, the lethality is 90.2%. Fermentation of all dilution concentrations inhibits the infection of the J2. After 30mg/kg ChA is added into the root of cucumber, the number of parasitized eggs is decreased by 63%, thereby showing that the ChA has a nematicidal activity and is biological nematicide with good application and development prospect.
Description
Technical field
The invention belongs to the microbial pesticide technical field.Be specifically related to a strain and have the chaetomium globosum of eelworm-killing activity, and the method for using its fungal metabolite biological control root knot nematode.
Background technology
The nematode of whole world serious harm plant has (Liu Weizhi, 2002) such as root knot nematode, Cyst nematode, aphelenchoides, Ditylenchus dipsaci, grain nematode and short body (root-rot) nematodes.Wherein, root knot nematode (Meloidogyne spp.) is as one of important pathogen biology that harms the crops, and giving all over the world, large-area farm crop have brought great harm (Zhao Hong etc., 2003).
The root knot nematode host range is very wide, surpasses 3000 kind of plant, belongs to 114 sections.The plants such as Solanaceae, Curcurbitaceae and Cruciferae, root knot nematode disease is more general.The life history of root knot nematode is fairly simple, becomes larva from egg development, casts off a skin through four times, becomes at last adult.Most of nematode life cycle is approximately 3-4 week.After root knot nematode laid eggs, ovum developed into first-instar young in chorion, and peeled once in chorion, be second instar larvae after the hatching, second instar larvae is perched in soil, waits for an opportunity to infect, usually invade in the root from the tip of a root, and in root, settle down and grow, become four-age larva through twice decortication again, before the 4th decortication, male larva becomes elongated shape, female larva is expanded and is long pyriform, after the last decortication, becomes respectively female, male imago.It is movable in soil that male worm leaves root, and female worm is stayed in the root, can lay eggs without mating, and ovum produces in the egg capsule of colloid.
In all root knot nematodes, the harm of Meloidogyne incognita (Meloidogyne incognita, or M. incognita) is the most serious.The annual crop production reduction loss that is caused by root knot nematode is greater than 1,000 hundred million dollars.Existing sterilant can not effectively suppress ever-increasing nematode population.And the traditional method of using in a large number the highly toxic pesticide such as monobromethane or Furadan to kill root knot nematode, owing to have environment and food safety risk, use progressively has been under an embargo.Therefore, seek their surrogate, such as biotechnological formulation and natural product, become study hotspot both domestic and external with effective harm that prevents and treats root knot nematode.Research finds that many fungies have potential nematicide activity.
Recently, ascomycetous fungus chaetomium globosum Chaetomium globosum(or C.globosum) begun to be applied to Strategies of Agricultural Bio-control, for example, as the biological prevention and control agent of plant pathogenic microorganisms and aphid.Also there are some to report that this bacterium has eelworm-killing activity.The rough nutrient solution of C.globosum can suppress the ovum hatching (Meyer etc., 2004) of M. incognita and soy bean cyst roundworm (Heterodera glycines).The fermented liquid of ball Chaetomium Chaetomium Ch1001 bacterial strain not only has significant lethal effect to M. incognita second instar larvae, can also obviously reduce the quantity of formation (Yan etc., 2011) of root knot.But the research of the natural product of isolation identification eelworm-killing activity is few from C.globosum.
Nematophagous fungi can produce secondary metabolite infecting or to kill nematode, and these natural compoundss are that tool is potential in order to the chemical insecticide of controlling nematode and the substitute of fumigant.In addition, consider to be prevalent in the soil, and can be used as the biotechnological formulation of control pathogenic micro-organism and insect such as aphid that exploring the nematode-killing vitality of C.globosum and extracting biotechnological formulation to become the from now on primary study of commercial and agricultural application.Domestic and foreign literature retrieval shows, at present also not with the secondary metabolite of the chaetomium globosum report as the control of nematode medicament.Therefore utilize the tunning of C.globosum to prepare the approach that nematocides is a worth exploration.
Summary of the invention
The objective of the invention is to select root knot nematode is had the fungal bacterial strain of high insecticidal activity, and with bacterial strain routinely after the liquid fermentation and culture, from meta-bolites, extract and have the effective constituent of nematocidal active material, exploitation wireworm-killing biologic agricultural chemicals, wherein, described root knot nematode Meloidogyne incognita preferably.
The present invention just is being based on above-mentioned theory, take root knot nematode as target, seeks efficient nematocidal active material from secondary fungus metabolite, intends seeking new resource into research and development new bio nematocides.In the process of seeking the potential GR of tool, we obtain a strain chaetomium globosum C.globosum NK106 at separation, and separation and purification goes out a kind of secondary metabolite chaetomium globosum element chaetoglobosin A(ChA from its fermented liquid).Further further investigation finds that the fermented liquid of this bacterial strain and the ChA of separation and purification have the very strong activity of killing to M. incognita.
The present invention at first provides a strain to have the fungal bacterial strain of eelworm-killing activity, be ascomycetous fungus chaetomium globosum Chaetomium globosum NK106, " China Committee for Culture Collection of Microorganisms's common micro-organisms " center " (address: Datun Road, Chaoyang District, Beijing City; Institute of Microorganism, Academia Sinica), culture presevation CGMCC6716 have been preserved on October 29th, 2012.C.globosum NK106 is the strain that filters out from a large amount of fungal bacterial strains has good eelworm-killing activity to Meloidogyne incognita fungal bacterial strain.
The present invention simultaneously from the fermented liquid of chaetomium globosum C.globosum NK106 separation and purification go out a kind of secondary metabolite chaetomium globosum element chaetoglobosin A(ChA).This meta-bolites ChA can be used in the control plant nematode, particularly M. incognita is had the very strong activity of killing.This metabolite is to be prepared by extraction after the liquid fermentation and culture of fungal bacterial strain process routine of the present invention.
Advantage of the present invention and positively effect:
The invention provides the chaetomium globosum C.globosum NK106 that a strain has eelworm-killing activity.
Fungal metabolite ChA provided by the invention is the material of finding to have first eelworm-killing activity both at home and abroad.
GR of the present invention is pure biotechnological formulation, can be used for the biological control to plant nematodes such as root knot nematodes.Nematocides of the present invention has no adverse effects for all biologies such as environment and plant, people, animals, and production cost is low, and production process is simple, and is free from environmental pollution.The microorganism nematocides does not have poisoning to cucumber in the experimental concentration scope, can promote and irritate on the contrary the growth of crop, and security is good.The present invention will be extremely important for the biological control of Plant nematode, the Application and Development of microorganism nematode killing agent and the nuisanceless production of agricultural-food.
Description of drawings
Fig. 1 is the thalli morphology of chaetomium globosum Chaetomium globosum NK106.A is the upper thalli morphology of cultivating for 2 weeks of solid PDA, and B is the thalli morphology of cultivating among the liquid PDA 9 days.
Fig. 2 is contained chaetomium globosum element ChA in high performance liquid chromatography (HPLC) the analytical procedure Purification and Characterization NK106 tunning.A is the ChA standard model, and appearance time 14.490min, B are the NK106 tunnings, appearance time 14.480min.
Fig. 3 is that different concns fungi fermentation filtrate and purification ChA are on the impact of J2 mortality ratio.A is the fermentation liquor treatment of different concns, and B is that the ChA of different concns processes.
Fig. 4 infects the impact of root to it after different concns fungi fermentation filtrate and purification ChA process nematode.A is the fermentation liquor treatment of different concns, and B is that the ChA of different concns processes.
Bacterial strain with eelworm-killing activity provided by the invention is the saprophytic ascomycetous fungus of a strain, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on October 29th, 2012, preservation address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, culture presevation CGMCC6716, Classification And Nomenclature: Chaetomium globosum NK106.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.These embodiment only are not used in for explanation the present invention and limit the scope of the invention.
The cultural characteristic of embodiment 1, chaetomium globosum C.globosum NK106 of the present invention
C.globosum NK106 strains separation of the present invention is on pine and cypress (Cupressns sp.) bark in somewhere, northeast.Bacterial strain on the PDA flat board 28 ℃ cultivated for 2 weeks after, thalline presents brown (shown in Figure 1A), collects spore with sterilized water and obtains spore suspension.Spore suspension is transferred in the 500ml flask that contains 200ml PDA potato substratum, and in 28 ℃, 9 days (as shown in Figure 1B) cultivated in the 180rpm concussion.Each flask inoculation 1 * 10
5Individual spore.PDA culture medium prescription (g/L): potato 200g, sucrose 20g adds in the agar 20g(solid medium).
Embodiment 2, C.globosum NK106 tunning treating processes provided by the invention, the analysis and identification of product
The fermented liquid of cultivating among the embodiment 1 is carried out suction filtration, so that mycelia is come with separation of fermentative broth in PDA; Gained fermented supernatant fluid behind the suction filtration is mixed extracted overnight with the equal-volume methylene dichloride; Slightly carried product after using rotatory evaporator concentrated to gained organic phase filtrate of upper step, carried out high performance liquid chromatography (HPLC) Analysis and Identification and collection.
HPLC selection analysis type C-18ODS chromatographic column (4.6 * 250mm, Kromasil), chromatographic condition: detect wavelength 227nm, flow velocity 1mL/min, column temperature are room temperatures, and moving phase is methyl alcohol: water=7:3.Show by experiment, contain the material (experimental result see Fig. 2) identical with ChA standard substance retention time in the tunning.(A.ChA standard substance, 14.490min; The B.NK106 tunning, 14.480min).According to going out peak area, the method for Application standard curvilinear regression can calculate the content of ChA in the tunning.ChA reaches maximum value 50.5mg/L when being cultured to the 9th day.NK106 can stably manufactured ChA in the PDA substratum.
The eelworm-killing activity of embodiment 3, NK106 bacterial strain fermentation liquor and purification ChA
The fermented liquid experiment is provided with 100%, 50%, 25%, 12.5% fungi filtrate (Filtrates) and PDA substratum (Medium, contrast) 5 processing, the ChA experiment is provided with 4 processing of 3 μ g/mL, 30 μ g/mL, 300 μ g/mL and 10% methyl alcohol (MeOH, contrast).Process all sterile distilled water (SDW) as blank for two groups.Each processing comprises 6 repetitions.Following experimental technique (2), (3) all use these two groups processing to test.
1. experimental technique
(1) preparation of nematode and ovum
Testing used worm's ovum, is that the root of the color green pepper (Capsicum annuum L.) of infecting from Meloidogyne incognita obtains.Be placed on after soil on the root cleaned in 1% the clorox to stir with agitator the ovum in the pieces of an egg is discharged, then cross 200 orders and 500 mesh sieves, collect ovum on the 500 purpose sieves.Second instar larvae J2 was then obtained by the ovum hatching in 2-3 days.
(2) fungi fermentation filtrate and purification ChA are on the impact of J2
With 24 orifice plates research NK106 ferment filtrate and the ChA lethality rate to Meloidogyne incognita J2.Nematode suspension is put into (comprising about 100 J2 among the 0.1mL) aperture of 24 orifice plates that contain different concns filtrate and ChA, all with sterilized water as negative contrast.Behind the inoculation nematode, 24 orifice plates are placed in 25 ℃ the incubator.In order to study fungi filtrate to the lethality rate of nematode, nematode is processed death toll and the sum of record nematode behind 24h, 48h and the 72h in endogenetic fungus filtrate, and the calculation correction mortality ratio.Stimulate the reaction of rear nematode to determine whether nematode is dead by observing NaOH.
(3) fungi fermentation filtrate and purification ChA infect the impact of root to it after processing nematode
Test is carried out in the Nankai University solarium.Heat treated soil (wherein comprises 0.74g K in the dry ground
2SO
4, 0.74gCO (NH
2)
2, 0.48g NH
4H
2PO
4) with after the vermiculite equal-volume mixes, put into the flowerpot of diameter 9cm.Plant cucumber in the seedling basin, be transplanted in the flowerpot after the week.After 4 days, after Meloidogyne incognita is processed 4h with different concns fungi fermentation filtrate and purification ChA, be inoculated in each flowerpot 1000, water a plant every day as required.Inoculate and gather in the crops plant after 9 days, slowly wash soil on the root with showerhead after, place the 50ml centrifuge tube, in centrifuge tube, put water logging and do not have plant root and in-20 ℃ refrigerator, place 24h.During separation, the root that from refrigerator, take out to freeze, after at room temperature dissolving plant root is placed on organize stir in the pulverizer 30s with discharge in the root tissue nematode and to nematodal accounting.
(4) purification ChA is on the impact of nematode breeding
With the soil plantation cucumber seedling of crossing with (3) same treatment, be transplanted in the flowerpot after the week.After 4 days, in flowerpot, add concentration and be 0.3,3, the purification ChA of 30mg/kg soil.After 2 days ovum is inoculated in the soil 1000 in each flowerpot.Behind the self-sow 35 days, results plant: measure plant height and the fresh weight of plant, then 105 ℃ of lower 0.5h in baking oven, then baking 10h under 80 ℃.The preparation method of root ovum, the root of results is cut into the segment of 1-2cm with scissors, then be placed on concentration and be in 1% the clorox, (sieve of top is 200 orders with sieving behind the A-88 tissue mashing refiner stirring 12min, following sieve is 500 orders), collect the dry weight of surveying root after with sprinkler head the root on 200 mesh sieves being rinsed well.Ovum is then stayed on 500 mesh sieves.In the centrifuge tube of 50ml, then last constant volume observes counting with inverted microscope to 40ml with ovum collecting.
2. experimental result
(1) NK106 bacterial strain fermentation liquor and purification ChA are on the impact of J2
After J2 was processed 24h, the fermented liquid of 12.5% concentration still had nematicide effect (Fig. 3 A).Lethality rate reaches maximum value 81.3% during the fermentation liquor treatment 72h of 100% concentration.Concentration is that mortality ratio reached 90.2%(LC50=126.5 μ g/ml after the ChA of 300 μ g/mL processed J248h) (Fig. 3 B), and nearly all nematode is all dead after 72h processes.Has significant difference between the mortality ratio that different concns is processed.
(3) NK106 bacterial strain fermentation liquor and purification ChA are on the impact of nematode infection
Compare with the PDA substratum of not inoculating bacterial classification with sterilized water, C.globosum NK106 fermented liquid can significance reduce infect (Fig. 4 A) of nematode.Fermented liquid and ChA show the rule that relies on concentration to infecting of nematode.Compare with negative contrast sterilized water, ChA can significance suppress the nematode infection cucumber, the 31.7%(Fig. 4 B during 59.4% and 30 μ g/mL concentration when inhibiting rate is respectively 300 μ g/mL concentration).Come to the same thing therewith, fermentation liquor treatment has also all reduced the quantity that infects of nematode.Compare with sterilized water, MeOH and PDA substratum all do not have impact to infecting.Infecting is M. incognita one of critical stage in the life history, and the infection ability of blocking-up nematode can illustrate that the nematicide of ChA is active.
(4) NK106 bacterial strain fermentation liquor and purification ChA are to nematode breeding and cucumber seedling affects on the growth
After processing 35 days, the ChA significance of 30mg/kg reduces the breeding of line eggs, has reduced compared with the control 63%, corresponding therewith, when 30mg/kg concentration was processed, the plant-growth amount significantly improved, the root biomass improves 3.4 times, and the biomass of stem improves 1.9 times (table 1).The result shows that ChA has eelworm-killing activity really.
Table 1 different concns ChA is to nematode breeding and cucumber seedling affects on the growth
The fermentation culture that the present invention has illustrated C.globosumNK106 has the characteristic of anti-Meloidogyne incognita (M. incognita).To J2 survive, infect, a series of inhibition such as breeding confirms that this epiphyte product ChA has strong eelworm-killing activity.As far as we know, this is to find that first ChA has the nematicide effect.This has determined ChA and can be combined with other plant pathogenic nematode administrative skill as a kind of control of nematode agent.Following practical application also needs field experiment and to the effect assessment of other plant pathogenic nematode.Simultaneously, NK106 is the fungi that a strain has using value, particularly to the nematocidal effect of Meloidogyne incognita, demonstrates its good application and development prospect.
Claims (5)
1. a strain has the fungal bacterial strain of eelworm-killing activity, ascomycetous fungus chaetomium globosum Chaetomium globosum NK106, and culture presevation number: CGMCC6716, Classification And Nomenclature are Chaetomium globosum.
2. bacterial strain according to claim 1 is characterized in that this bacterial strain production has the secondary metabolite chaetomium globosum element chaetoglobosin A(ChA of nematocidal effect).
3. a mematocide is characterized in that, comprises the fungi claimed in claim 1 of significant quantity or fungal metabolite claimed in claim 2 as activeconstituents.
4. fungi claimed in claim 1 or fungal metabolite claimed in claim 2 or the application of mematocide claimed in claim 3 in the plant nematode biological control.
5. application according to claim 4 is characterized in that, described plant nematode is root knot nematode (Meloidogyne spp.).
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| CN107721908A (en) * | 2017-09-21 | 2018-02-23 | 黄河科技学院 | A kind of method that chaetomium globosum A precursor compounds are extracted from chaetomium globosum |
| CN110169967A (en) * | 2019-05-15 | 2019-08-27 | 三峡大学 | Chaetomium globosum A is as the application on tyrosinase inhibitor |
| CN111944705A (en) * | 2020-09-02 | 2020-11-17 | 鲁东大学 | Solid-state fermentation method of chaetomium globosum, prepared microbial inoculum and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107721908A (en) * | 2017-09-21 | 2018-02-23 | 黄河科技学院 | A kind of method that chaetomium globosum A precursor compounds are extracted from chaetomium globosum |
| CN110169967A (en) * | 2019-05-15 | 2019-08-27 | 三峡大学 | Chaetomium globosum A is as the application on tyrosinase inhibitor |
| CN111944705A (en) * | 2020-09-02 | 2020-11-17 | 鲁东大学 | Solid-state fermentation method of chaetomium globosum, prepared microbial inoculum and application thereof |
| CN111944705B (en) * | 2020-09-02 | 2022-03-22 | 鲁东大学 | Solid-state fermentation method of chaetomium globosum, prepared microbial inoculum and application thereof |
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