CN103409324B - Trichoderma viridescens for preventing and treating pepper phytophthora blight and compound thereof - Google Patents
Trichoderma viridescens for preventing and treating pepper phytophthora blight and compound thereof Download PDFInfo
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- CN103409324B CN103409324B CN201310264340.0A CN201310264340A CN103409324B CN 103409324 B CN103409324 B CN 103409324B CN 201310264340 A CN201310264340 A CN 201310264340A CN 103409324 B CN103409324 B CN 103409324B
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Abstract
The invention discloses trichoderma viridescens for preventing and treating pepper phytophthora blight and a compound thereof. The trichoderma strain is named gradually green trichoderma viridescens TS0404, and has a Preservation No. of CGMCC NO. 7821. The structural formula of the compound is shown in Figure 10. According to the invention, gradually green Trichoderma obtained through separating and screening has a strong inhibitory effect on phytophthora capsici, and has a good biocontrol potential to phytophthora capsici. According to the invention, Trichoderma viridescens with an antagonistic effect on phytophthora capsici is screened from a large number of trichoderma viridescens, a secondary metabolic compound '6-pentyl-2H-pyran-2-ketone' is separated and purified from secondary metabolites of the trichoderma viridescens TS0404, and the compound has a strong inhibitory activity to a phytopathogen Phytophthora capsici of oomycetes.
Description
Technical field
The present invention relates to technical field of biological control, particularly relating to a kind of trichoderma strain for preventing and treating capsicum epidemic disease and compound.
Background technology
Capsicum epidemic disease is commonly called as " dead seedling is sick ", is a kind of soil-borne disease caused by Phytophthora capsici (Phytophthora capsici).Capsicum epidemic disease is a kind of destructive disease on China capsicum, can cause serious production loss.Conventional Control Technology has been difficult to effective control, and easily causes ecology, environmental problem.
How effectively to prevent and treat capsicum epidemic disease is the problem of producing and research being paid close attention to always.In breeding for disease resistance, do not realize real breakthrough technically so far, on producing, there is no anti-capsicum epidemic disease kind truly.At present, main agricultural cultivation measure control and the chemical pesticide control of adopting is prevented and treated capsicum epidemic disease.Agricultural cultivation measure comprises the cultivation technique of prevention pathogenic bacteria propagation as drip irrigation, high ridge and overlay film etc., but these cultivation techniques reduce pathogenic bacteria for target, disease is once occur, agricultural cultivation measure can not effectively control the popular of disease rapidly, therefore, chemical pesticide remains the prophylactico-therapeutic measures of main flow.
Along with people are to the growing interest of the problems such as agricultural sustainable development, living environment, food safety and pathogenic bacteria resistance, the importance of biological control is more outstanding, utilize the biocontrol microorganisms with disease control effect, be made into biotechnological formulation for the production of upper controlling disease.In recent years, the biocontrol microorganisms prevented and treated around capsicum epidemic disease mainly concentrates on Trichoderma (Trichoderma spp.) and some bacteriums.
Publication number is that the Chinese patent literature of 102719364A discloses a strain trichoderma harzianum strain and the application in prevention and control capsicum epidemic disease thereof.Trichoderma harziarum (Trichoderma harzianum) HNA-12 that this patent provides, preserving number is CGMCC No.5989, although this trichoderma harziarum HNA-12 can suppress pathogenic bacteria to a certain extent, promote pepper plant growth, prevention and control pepper plant generation capsicum epidemic disease, poor to the restraining effect of Phytophthora capsici.
The Biocontrol Mechanism of Trichoderma generally has four kinds, the bacteriolysis of Competition, hyperparasitism, enzyme and the generation of Antibiotics.Wood is mould can produce the multiple secondary metabolite with antibacterial effect, and the antibiotics of the mould generation of most kind wood is also more than a kind of.Wooden mould distribution range is extremely wide, and under different environmental stimuluses, wood is mould by different metabolic processes, can produce various secondary metabolites.These secondary metabolites, for environmental facies to gentleness, therefore, the metabolite having bacteriostatic activity from the mould middle extraction of wood is a kind of important means of biological control, is also the basis of chemical bactericide synthesis.
At present, many reports about being prevented and treated pathogenic bacteria by the mould secondary metabolite of wood have been had: the inhibiting rate of simple pyranone (Simple pyrones) material 6-amyl group-pyrone Rhizoctonia solani (Rhizoctonia solani) purified from the mould metabolite of wood reaches 69.6%.Glue green trichoderma toxin is the one in diketopiperazine class material, and research finds that glue green trichoderma toxin is to Pythium ultimum, has reasonable restraining effect.But, the research of current metabolite great majority for be the higher fungies such as ascomycetes, basidiomycetes, fewer to relate to low researchs waiting oomycetes such as epidemic disease are mould.
Summary of the invention
The invention provides a strain wood mould, to capsicum epidemic disease, there is good Biocontrol Potential.
One strain wood is mould, Classification And Nomenclature is gradually green trichoderma (Trichoderma viridescens), complete called after is green trichoderma (Trichoderma viridescens) TS0404 gradually, this bacterial strain is kept at the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on June 27th, 2013, preserving number is: CGMCC NO.7821.
Bacterial strain of the present invention 25 DEG C of cultivations, 5 days bacterium colonies on CMD substratum can cover whole flat board, bacterium colony is thin, white mycelium, the radial loose growth of mycelia, colony edge position aerial hyphae is thickening gradually, and the edge of wide strip becomes fine hair shape, conidiophore main shaft is high-visible, approximate 90 ° of side shoot crotch angle, conidium Asia is spherical, and bottle stalk has prolification.
Present invention also offers the mould application in control capsicum epidemic disease of described wood.
The mould metabolite secreted in its metabolic process of wood is one of main mechanism of control plant fungal pathogenic bacteria, experiment shows, trichoderma strain of the present invention can produce has inhibiting secondary metabolite to Phytophthora capsici bacteria growing, and inhibition is strong, having shown trichoderma strain of the present invention is Microbial resources with better Biocontrol Potential.
Described application specifically comprises:
(1) biological prevention and control agent is made by mould for described wood;
(2) after capsicum field planting, described biological prevention and control agent is imposed on Hot Pepper Seedling root.
The preparation method of described biological prevention and control agent comprises: prepare wooden mould seed liquor; Mould for described wood seed liquor is inoculated in the wheat boiled, cultivates 15 ~ 20 days, obtained described biological prevention and control agent.
The viable bacteria concentration of the mould seed liquor of described wood is 10
7~ 10
8individual/ml, is preferably 10
8individual/ml.
In every kilogram of wheat, the inoculum size of wooden mould seed liquor is 2 ~ 4ml, is preferably 3ml.
For making the mould full and uniform growth on wheat of wood produce spore, in culturing process, stir wheat in time.
The temperature of described cultivation is 22 ~ 27 DEG C, is preferably 25 DEG C.
The time of described cultivation is preferably 18 ~ 20 days, is more preferably 18 days.
When using, the consumption of described biological prevention and control agent is 1.5 ~ 2g/ strain.
Present invention also offers a kind of compound, there is following structural formula:
Present invention also offers the application of described compound in control plant Oomycete disease.
Described plant Oomycete disease is specially capsicum epidemic disease.
The pathogenic bacteria of capsicum epidemic disease is Phytophthora capsici (P.capsici), phytophthora blight of pepper is that a kind of soil passes oomycetes, survive the winter in soil invalid body with oospore or chlamydospore, experiment proves, described compound has bacteriostatic action, strong to the restraining effect of Phytophthora capsici, therefore, using this compound as activeconstituents, biological bactericide can be prepared, for the control of capsicum epidemic disease.
Compared with prior art, beneficial effect of the present invention is:
(1) the gradually green trichoderma that obtains of separation screening of the present invention, strong to the restraining effect of Phytophthora capsici, there is good capsicum epidemic disease Biocontrol Potential.
(2) the present invention screens trichoderma strain phytophthora blight of pepper to antagonistic action from a large amount of trichoderma strain, and separation and purification obtains compound " 6-pentyl-2H-pyran-2-one " from the secondary metabolite of trichoderma strain TS0404, this compound has strong inhibit activities to oomycetes phytopathogen Phytophthora capsici.
Accompanying drawing explanation
Fig. 1 is cultivation after 7 days, slightly puies forward the inhibition of metabolite to Phytophthora capsici;
Fig. 2 is cultivation after 7 days, slightly puies forward the inhibition of metabolite to persimmon anthrax-bacilus;
Fig. 3 is the result of component 3 after thin-layer chromatography;
Fig. 4 is the inhibition of component 3 pairs of phytophthora blight of pepper;
Fig. 5 be in component 3 the 2nd spot to the inhibition of phytophthora blight of pepper;
Fig. 6 is the infrared spectrogram of the spot 2 in component 3;
Fig. 7 is the hydrogen spectrogram of the spot 2 in component 3;
Fig. 8 is the carbon spectrogram of the spot 2 in component 3;
Fig. 9 is Dept135 ° and 90 ° of collection of illustrative plates of the spot 2 in component 3;
Figure 10 is the spot 2 compound structure figure in component 3.
Embodiment
Below in conjunction with specific embodiment, the present invention is further explained.
The separation andpreconcentration of embodiment 1 trichoderma strain
1, the separation of trichoderma strain
From Zhangjiajie, Mount Taibai and Mount Taishan gathers soil sample.Soil sample sampling depth is for fallen leaves are lower or earth's surface about 5cm, and load in aseptic plastic bag after gathering and seal, take back laboratory and be separated, soil sample to be separated is stored in 4 DEG C of refrigerators.After soil sample fully being mixed, the grogs that takes a morsel is put on the PDA containing paraxin, every ware 4 parts of grogs, repeats 3 times.Be placed in 25 DEG C of incubator dark culturing, after wooden mould product spore, timely picking spore proceeds in new PDA flat board, and dark culturing at 25 DEG C also obtains pure growth.
2, the qualification of trichoderma strain
1) identification of morphology
(1) colony characteristics is observed: colony characteristics is observed and carried out on CMD substratum, its method cuts bacterium cake 1 piece with punch tool (diameter 5mm) from the colony edge of CMD active growth, be inoculated into culture dish (diameter 9cm) the 1cm place, edge containing 20mlCMD, 20 DEG C of alternation of light and darkness cultivate visual inspection colony characteristics after 7-14 days.
(2) observation of conidiophore: the microscopic examination that conidium, conidiophore and bottle obstruct, that cultivation wood is mould on CMD substratum, its method cuts bacterium cake 1 piece with punch tool (diameter 5mm) from the colony edge of CMD active growth, be inoculated into culture dish (diameter 9cm) the 1cm place, edge containing 20ml CMD, 20 DEG C of alternation of light and darkness are cultivated, when on bacterium colony, just formation conidiophore and bottle obstruct time (3-4 days), the form of microscopy observation conidiophore and bottle stalk and size.Cultivate after 7-14 days, under 100 times of oily mirrors, observe conidial shape and size.
The upper 25 DEG C of cultivations of interpretation of result: CMD 5 days bacterium colonies can cover whole flat board, and bacterium colony is thin, white mycelium, the radial loose growth of mycelia, and colony edge position aerial hyphae is thickening gradually, and the edge of wide strip becomes fine hair shape.Conidiophore main shaft is high-visible, approximate 90 ° of side shoot crotch angle.Conidium Asia is spherical, and bottle stalk has prolification.
2) Molecular Identification
(1) CTAB method is adopted to extract the genome of trichoderma strain.
(2) pcr amplification of ITS rDNA, Tef1-α and rpb2
ITS1-5.8S-ITS2 fragment adopts fungi Internal Transcribed Spacer universal primer ITS6 and ITS4 amplification ITS and 5.8S rDNA(Cooke & Duncan1997).
Tef1-α gene adopts universal primer Ef728M(Carbone and Kohn1999) and tef1R(Kullnig-Gradinger et al.2002).
Set up PCR reaction system (table 1), PCR primer is detected at 1.2% agarose gel electrophoresis, and carry out photographic analysis in gel imaging system.After observing suitable ITS band, be that PCR primer reclaims kits PCR primer with adsorption column, ITS, tef1-α, rpb2 amplified production carries out two-way order-checking with amplimer respectively.
The connection that sequence Clustal X program after order-checking carries out rDNA sequence is joined (alignment) and is compared, edits, and then submits Gen Bank to.ITS1, ITS2 and 5.8S rDNA sequence mould for wood in sequence and Gen Bank is carried out tetraploid rice.
Table 1PCR reaction system (50 μ l)
Interpretation of result:
The morphological specificity of bacterial strain TS0404:
The upper 25 DEG C of cultivations of CMD 5 days bacterium colonies can cover whole flat board, and bacterium colony is thin, white mycelium, the radial loose growth of mycelia, and colony edge position aerial hyphae is thickening gradually, and the edge of wide strip becomes fine hair shape.Conidiophore main shaft is high-visible, approximate 90 ° of side shoot crotch angle.Conidium Asia is spherical, and bottle stalk has prolification.
The ITS sequence of bacterial strain TS0404 is as shown in SEQ ID NO.1, and Tef sequence is as shown in SEQ ID NO.2, and Rpb2 sequence is as shown in SEQ ID NO.3.
According to morphology and molecular systematics qualification result, bacterial strain TS0404 is gradually green trichoderma (Trichoderma viridescens), called after is green trichoderma (Trichoderma viridescens) TS0404 gradually, this bacterial strain has been kept at the China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) being positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, deposit number is: CGMCC NO.7821, and preservation date is on June 27th, 2013.
The screening of embodiment 2 antagonistic Trichoderma bacterial strain
With embodiment 1 the trichoderma strain that obtains of separation for strains tested.
(1) glassine paper method
Whether can produce with glassine paper culture method detection trichoderma strain and have inhibiting secondary metabolite to Phytophthora capsici bacteria growing.Glassine paper is cut into the disk of diameter 9cm, separates glassine paper disk respectively with the scraps of paper, parcel after superposition, high-temperature sterilization.Before use, glassine paper disk is placed in the container containing aqua sterilisa, makes it moistening.PDA is poured in the sterilizing culture dish of diameter 9cm, to be cooled solidify after aseptically sterilizing glassine paper is attached to media surface, volatilize excessive moisture in Bechtop.
Trichoderma strain and the switching of Phytophthora capsici bacterial strain are activated on PDA substratum, after grown 48h, uses diameter 5mm: punch tool is cut into bacterium cake at colony edge.By pure culture biscuits involvng inoculation to the culture dish central authorities containing glassine paper.Cultivate in incubator under 25 DEG C of dark conditions.Until wood is mould cover with culture dish soon time, throw off glassine paper, and the bacterium cake of the upper phytophthora blight of pepper of inoculation.Each process in triplicate, is cultivated in 25 DEG C of incubators, observes the upgrowth situation that epidemic disease is mould.
(2) crude extract method
The trichoderma strain with metabolite activity that early stage screens is carried out the experiment of metabolite crude extract, determines its metabolite activity further.
Be seeded in by the trichoderma strain of activation in the culture dish of cover glass paper, each bacterium connects 20 culture dish.After bacterium colony covers with glassine paper, move Trichoderma fall and glassine paper.Draw broken substratum with clean scalpel, be extracted with ethyl acetate 3 times, combining extraction liquid is (40 DEG C) concentrated evaporate to dryness on a rotary evaporator, adds 2ml acetone and is again dissolved by extract.In sterilized culture dish, pour the PDA substratum 10ml of thawing into, cool in Bechtop and vapor away unnecessary moisture.Draw the middle that 20 microlitre crude extracts are applied to substratum, then the bacterium cake (5mm) of Phytophthora capsici is placed on crude extract and (makes mycelia contact crude extract).Cultivate under 25 DEG C of dark conditions, observe every day and record the mould upgrowth situation of epidemic disease.Only to add 20 L acetone for contrast.Each process in triplicate.
Interpretation of result
To Zhangjiajie, all bacterial strains that Mount Taibai is separated with Mount Taishan screen, and obtain the growth that the mould metabolite of 34 strain wood can suppress Phytophthora capsici, the mould growth of epidemic disease is less than bacterial strain 75 strain of contrast epidemic disease mould 1/2.
Can produce for 34 strains that the wood of active metabolite is mould carries out crude extract determination of activity experiment, found that gradually green trichoderma bacterial strain (Trichoderma viridescens) is numbered TS0404(and pick up from Mount Taibai) crude extract can suppress the growth of Phytophthora capsici completely.
Wood is mould secretes various lytic enzyme as chitinase (chintinases) in its metabolic process, dextranase (glucnaases) and proteolytic enzyme (proteinases), the cell walls of their energy degradative fungis is one of main mechanism of control plant fungal pathogenic bacteria.Glassine paper can pass through the albumen of at least 90Dka, and these lytic enzymes enter (Kullniget.al, 2000) in nutrient agar by glassine paper.Therefore, the metabolite entered in nutrient agar by glassine paper not only may contain various albumen (lytic enzyme, antibacterial peptide), also containing antimicrobial compounds and other secondary metabolite, thus produces the effect suppressing Phytophthora capsici bacteria growing.
The application of embodiment 3TS0404 trichoderma strain in capsicum epidemic disease control
The preparation of biological prevention and control agent
(1) wooden mould TS0404 spore suspension is prepared
Mould for wood TS0404 is seeded in PDA substratum, cultivates 6 days at 25 DEG C; After cultivation completes, scraped by spore in aqua sterilisa, preparation obtains TS0404 spore suspension, and (viable bacteria concentration is 10
8individual/mL).
(2) Trichoderma preparation is prepared
Mould for wood TS0404 spore suspension is inoculated in the wheat (feed wheat) boiling sterilizing, needed for every kilogram of wheat, the consumption of wooden mould TS0404 spore suspension is stir in time between 3mL incubation period, at 12 h light and 12 h dark alternate cultures after 18 days, substratum produces a large amount of spores, spore is Trichoderma preparation together with wheat.
Biological and ecological methods to prevent plant disease, pests, and erosion is tested
Adopt the main cultivation in Zhejiang area capsicum susceptible variety Hangzhoupro green pepper No. 2, carry out biological and ecological methods to prevent plant disease, pests, and erosion test.
(1) after capsicum field planting (namely transplanting seedlings), around rhizosphere, use Trichoderma preparation, wherein, the amount of application of Trichoderma preparation is the every strain of 2g/.
After (2) one months, with root-pouring method inoculation phytophthora blight of pepper spore suspension, spore concentration is 10
8individual/mL, every strain capsicum inoculation 3ml.Each process 20 strain, designs three repetitions.
(3) inoculate after pathogenic bacteria after 22 days, record incidence, calculates prevention effect.
Prevention effect (%)=100 × (contrast disease index-process disease index)/(contrast disease index)
Through statistics, Trichoderma preparation of the present invention is 72.5% to the prevention effect of Phytophthora capsici.
The extracting and developing of embodiment 4 metabolite, determination of activity
One, experimental technique
1, the cultivation that wood is mould
(1) solid culture and extracting method
The trichoderma strain TS0404 of activation is seeded in the large culture dish of the diameter 12cm of cover glass paper, Dual culture 500 ware, cultivates 15 days under 25 DEG C of dark conditions, remove glassine paper.Draw broken substratum with clean scalpel, be extracted with ethyl acetate 3 times, enriched material with evaporate to dryness concentrated at 40 DEG C, dissolves with a small amount of acetone, pipettes solution for subsequent use to centrifuge tube with glass pipette by combining extraction liquid on a rotary evaporator again.
(2) liquid culture and extracting method
The trichoderma strain TS0404 of activation is inoculated into containing 200ml PDB(potato dextrose broth) in the 500ml triangular flask of nutrient solution, in shaking table, under 23 DEG C of dark conditions, 140r/min cultivates 15 days.By nutrient solution filter paper filtering, filtrate, with the extraction into ethyl acetate 2 times of 1/2 volume, collects bacterium liquid 40L altogether, and extraction liquid is merged the concentrated dry product that slightly got sample.
2, the chromatography purification of metabolite and the determination of activity of different components
(1) column chromatography
After being merged by the crude extract collected, carry out column chromatography for separation test.
Silica gel pre-treatment: after silica gel (200-300 order) is washed respectively with diluted acid, diluted alkaline, be washed with distilled water to neutrality, again silica gel is suspended in 95% ethanol, hold over night, pour out ethanol, then repeat once, suction filtration, after flinging to ethanol, be put in moisture eliminator dry 8h at 110 DEG C after and save backup.
Sample adsorption: take a morsel column chromatography silica gel in culture dish, adds the crude extract with acetone solution, fully stirs, crude extract is fully mixed with silica gel, uniform adsorption.Leave standstill for some time, after acetone volatilization completely, for subsequent use.
Dress post loading: the silica gel (200-300 order, column chromatography is special) taking example weight 100 times, adds appropriate sherwood oil, agitation as appropriate, silica gel and sherwood oil are mixed, slowly pours in pillar, in process, raps pillar to remove issuable bubble.The piston opened below pillar flows out by eluent.When eluent liquid level is close to silica gel aspect, silica gel adds a cotton, then must be sprinkling upon even for the silica gel adsorbing crude extract on cotton, finally add an absorbent cotton again in the superiors, in order to avoid destroy Silica Surface when adding elutriant.
Cross post to collect: be made into gradient elution agent by sherwood oil and ethyl acetate, ratio is respectively 10:1,9:1,8:1 ... until 1:1.Normal pressure drip washing, collects elutriant with sample hose, and every 5ml collects a pipe.The elutriant collected in each bottle is carried out thin-layer chromatography, observes its speckle displacement, elutriant identical for speckle displacement is merged.
Activity experiment: after the component collected is merged, by after elutriant evaporate to dryness in Rotary Evaporators, add a small amount of acetone solution, transfer in sample hose, carry out activity experiment respectively.In sterilized culture dish, pour the PDA substratum 10ml of thawing into, cool in Bechtop and vapor away unnecessary moisture.Draw the middle that 20 microlitre crude extracts are applied to substratum, the bacterium cake of Phytophthora capsici is placed on crude extract and (makes mycelia contact crude extract).Cultivate under 25 DEG C of dark conditions, observe every day and record the mould upgrowth situation of epidemic disease.Only to add 20 L acetone for contrast.Each process in triplicate.
(2) thin-layer chromatography
The preparation of thin layer chromatography board: 50g tlc silica gel (GF254) is mixed with suitable ratio with water, abundant stirring, adds a small amount of Xylo-Mucine as tamanori, furnishing pasty state, leave standstill after 3 hours, remove bubble with ultrasonic washing instrument, it evenly must to be layered on clean sheet glass, thickness is about 0.5mm, at room temperature dry, in the baking oven of 110 DEG C, activate 1 hour, after treating that its temperature is down to room temperature, silica-gel plate is transferred in moisture eliminator for subsequent use.
Point sample: in order to reduce the impact of impurity in silica gel, first with developping agent, pre-deployment is carried out to remove the impurity in silica gel to the silica-gel plate prepared before point sample, at 105 DEG C, 30min is activated again after being dried by the silica-gel plate of pre-deployment, in moisture eliminator, be cooled to room temperature, take a morsel crude extract sample, uses a small amount of acetic acid ethyl dissolution, silica-gel plate carries out thin-layer chromatography (Thin-layer chromatography, TLC) analyze, 1.5cm at the bottom of point of sample distance plate, dries up with blower.
Launch: the excellent silica-gel plate of point is placed in the chromatography cylinder using developping agent saturated in advance, launches with upright ascending method, and record expansion situation.Developping agent is the mixture of sherwood oil and ethyl acetate, and its ratio is about sherwood oil: ethyl acetate=4:1.
Silica-gel plate observes spot under 254nm uv analyzer, then fumigates with iodine vapor, determines speckle displacement.Remaining extraction sample is all placed on silica-gel plate and carries out chromatography, with Pencil marks spot profile.After sample spot scrapes respectively, silica gel powder adds acetone, is again dissolved by the fungal metabolite be adsorbed on silica-gel powder, repeat extraction 3 times, after united extraction liquid, with filter paper filtering, add a small amount of acetone after concentrated evaporate to dryness on a rotary evaporator extract is dissolved again, move in sample hose for subsequent use.
The sample solution of the different components obtained by thin-layer chromatography carries out activity test respectively.Activity experiment method is with crude extract activity experiment method.Take acetone as contrast, whole experiment in triplicate.
3, the Structural Identification of spot 2 in component three
Infrared spectroscopy: the spot 2 in component three is carried out Infrared spectroscopy;
Nuclear magnetic resonance spectroscopy: the spot 2 in component three is carried out Infrared spectroscopy.
Two, results and analysis
1, metabolite slightly gets sample the column chromatography for separation result of product
Extract solid culture ware and the liquid fermentation liquid of mass propgation by ethyl acetate, altogether obtain crude extract 5g.
Utilize silica gel by polarity by weak extremely strong order, with different solvent systems gradient elutions, to the elutriant collected respectively under uv analyzer and observe the position of spot in iodine cylinder, elutriant identical for speckle displacement is merged, the elutriant that spot intersects is rejected, and altogether obtains the elutriant of 5 components.Collect by equivalent, altogether collect 120 bottles of elutriants.Wherein, the ratio of first component elutriant is sherwood oil: ethyl acetate=10:1, and this ratio elutriant collects 15 bottles altogether.The ratio of second component elutriant is sherwood oil: ethyl acetate=8:1, and this ratio elutriant collects 10 bottles altogether.The ratio of the elutriant of the 3rd component is sherwood oil: ethyl acetate=6:1, collects 30 bottles altogether.The elutriant ratio of the 4th component is sherwood oil: ethyl acetate=4:1, collects 30 bottles altogether, and the elutriant ratio of the 5th component is sherwood oil: ethyl acetate=2:1, collects 35 bottles altogether.
As shown in Figure 4, carry out Antibacterial Activity to Phytophthora capsici respectively to being separated 5 components obtained, result shows, and component three has the activity that Phytophthora capsici mycelia can be suppressed to grow.
2, metabolite slightly gets sample the active testing result of product
As shown in Figure 1, the crude extract metabolite of trichoderma strain TS0404 shows the exercising result of Phytophthora capsici mycelia, and after 7 days, Phytophthora capsici bacterium colony cannot grow, and the mould normal growth of the epidemic disease contrasting (acetone is as blank), bacterium colony covers with whole culture dish.This illustrates that wood is mould and slightly carries in metabolite containing the activeconstituents suppressing Phytophthora capsici bacteria growing.
3, for the active testing result of anthrax-bacilus
As shown in Figure 2, the crude extract metabolite of trichoderma strain TS0404 shows the exercising result of persimmon anthrax-bacilus, and after 7 days, persimmon anthrax-bacilus bacterium colony cannot grow, and the mould normal growth of the epidemic disease contrasting (acetone is as blank), bacterium colony covers with whole culture dish.This illustrates that wood is mould and slightly carries in metabolite containing the activeconstituents suppressing the growth of persimmon anthrax-bacilus.
4, metabolite thin-layer chromatography and active testing result
The 3rd component collected by column chromatography carries out thin-layer chromatography expansion, the ratio of the developping agent used is sherwood oil: ethyl acetate=4:1, after silica-gel plate is taken out, after drying up developping agent with blower, be under the ultraviolet lamp of 254nm at wavelength, as Fig. 3, two spots can be observed, and after iodine is stifling, find no newly-increased spot.Rf value=0.62 of spot 2 and, this spot colors is denseer, and area is comparatively large, thinks that the compounds content of this spot is larger.Rf value=0.28 of spot 1.
After 2 spots obtained by component 3 thin-layer chromatography reclaim, use acetic acid ethyl dissolution compound respectively, by ethyl acetate evaporate to dryness in Rotary Evaporators, add a small amount of acetone solution compound, carry out Phytophthora capsici bacteriostatic activity test, as Fig. 5, result shows, spot 2 in component 3 can suppress the growth of Phytophthora capsici, and spot 1 does not have the bacteriostatic activity of Phytophthora capsici.Be separated through thin-layer chromatography, the amount finally obtaining spot 2 in component 3 is 0.5g.
5, spot 2 compound structure detected result:
Infrared spectra is 1738
-1there is the C=O absorption band that strong, 1634-1 and 1559
-1two C=C elongated strap.Think a-pyrone ring (a-pyrone ring) (see figure 6).Mass spectrum shows a parent ion at m/e166.Molecular formula is C
10h
14o
2, exact mass:166.10, molecular weight:166.22, m/z:166.10(100%), 167.10(10.9%), Elemental analysis:C, 72.26; H, 8.49; O, 19.25, the structural formula of this compound is as shown in Figure 10.Nucleus magnetic resonance (NMR)
1h signal is presented at 0.94(3, triplet), 1.4(6H, complex multiplet) 2.48(2H, triplet) be all unanimously to pentyl (see figure 7).Nucleus magnetic resonance (NMR)
13c Signal aspects at δ 12.95,21.99,26.37,30.84,33.13,102.92,112.16,145.03,163.75 and 166.81(see Fig. 8).Dept shows the adjacent H element number (see figure 9) of C.The analysis of IR and NMR collection of illustrative plates discloses, and this compound is completely the same with " 6-pentyl-2H-pyran-2-one " (the 6-pentyl-α-pyrone) reported.
This compound is by Collins and Halim(1972) find in T.viride nutrient solution for the first time and identify this compound.Since then, at T.harzianum(Claydon et al.1987) and T.koningii(Simon et al.1988) middle period obtains this compound.This metabolite has significant biological activity to basidiomycetes dry thread Pyrenomycetes (Rhizoctonia solani) and ascomycetes tomato fusarium oxysporum (Fusarium oxysporum f.sp.Lycopersici).This compound is also for preventing and treating the storage period Kiwifruit canker (Poole et al.1998) that botrytis cinerea (Botrytis cinerea) causes simultaneously.But also this compound does not have the research report of antagonistic action to oomycetes phytopathogen so far.But because oomycetes monoid belongs to Zao Wu circle, and basidiomycetes and arthroderma are in mycota, and this compound of Late Cambrian of the present invention has strong inhibit activities to oomycetes phytopathogen Phytophthora capsici.The main component of oomycetes cell walls is Mierocrystalline cellulose, and the main component being different from the cell walls of fungi is chitin.The present invention expands the frontier of this compound application, also for biological bactericide synthetic provides important information.
The activation analysis of embodiment 5 compound
With acetone diluted 6-pentyl-2H-pyran-2-one, be made into 100ppm respectively, the solution of 50ppm, 20ppm, 10ppm, 5ppm.Get the solution that 20 μ l dilute respectively, drip in the middle part of PDA flat board, the bacterium sheet (5mm) of placement containing phytophthora blight of pepper is on drop.Put culture dish to cultivate under 25 DEG C of dark conditions, to drip acetone soln for contrast, observe the growing state of Phytophthora capsici mycelia, statistics after 7 days.
6-pentyl-2H-the pyran-2-one of table 2 different concns is to the inhibition of phytophthora blight of pepper
Observations shows, when this compound concentration is more than 20ppm, can suppress the growth of Phytophthora capsici mycelia completely; During lower than 20ppm, grow the suppression (see table 2) be subject in various degree.
Claims (4)
1. a strain wood is mould, it is characterized in that, called after is green trichoderma (Trichoderma viridescens) TS0404 gradually, and deposit number is CGMCC NO.7821.
2. the mould application in control capsicum epidemic disease of wood as claimed in claim 1.
3. apply as claimed in claim 2, it is characterized in that, comprising:
(1) biological prevention and control agent is made by mould for described wood;
(2) after capsicum field planting, described biological prevention and control agent is imposed on Hot Pepper Seedling root.
4. apply as claimed in claim 3, it is characterized in that, the preparation method of described biological prevention and control agent comprises: prepare wooden mould seed liquor; Mould for described wood seed liquor is inoculated in the wheat boiled, cultivates 15 ~ 20 days, obtained described biological prevention and control agent.
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| CN103053624A (en) * | 2012-12-27 | 2013-04-24 | 浙江大学 | Method for control of phytophthora blight of pepper by mixed application of trichoderma preparation and fungicides |
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| CN103053624A (en) * | 2012-12-27 | 2013-04-24 | 浙江大学 | Method for control of phytophthora blight of pepper by mixed application of trichoderma preparation and fungicides |
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