CN102174416B - Angelica sinensis endophytic fungi Fusella sp DG09 with plant pathogenic bacteria resistant activity and fermentation solution and application thereof - Google Patents
Angelica sinensis endophytic fungi Fusella sp DG09 with plant pathogenic bacteria resistant activity and fermentation solution and application thereof Download PDFInfo
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Abstract
The invention discloses angelica sinensis endophytic fungi Fusella sp DG09 with plant pathogenic bacteria resistant activity and fermentation solution and application thereof. The Fusella sp DG09 was collected in China General Microbiological Culture Collection Center on January 7th, 2011, and has the collection number of CGMCC No.4543. The fermentation solution of the Fusella sp DG09 is prepared by the following method of: inoculating slant strains of the Fusella sp DG09 to a seed culture medium, performing shaking culture till an exponential growth phase, inoculating 10 percent of the cultured strains to a fermentation medium, culturing the strains for 4 to 5 days, performing centrifugation after the fermentation is finished to obtain supernate, and concentrating the supernate to obtain the fermentation solution. By numerous experiments, the endophytic fungi Fusella sp DG09 with strong plant pathogenic bacteria resistant activity of resisting gibberella saubinetti, early tomato phytophthora, tomato grey mould, rice sheath blight and the like is screened from angelica sinensis; and the endophytic fungi Fusella sp DG09 has good application prospect in biological prevention and control of plant diseases, and has important guidance significance for culture, production and sustainable utilization of medicinal materials such as the angelica sinensis and the like.
Description
Technical field
The present invention relates to the biologic product technology field, be specifically related to a kind of the have active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09, Fusella sp DG09 fermented liquid.
Background technology
When the dry root that is classified as umbelliferae angelica Angelica sinensis (Oliv.) Diels, it is sweet, hot, warm to distinguish the flavor of, and returns liver, the heart, the spleen channel, and having enriches blood invigorates blood circulation, menstruction regulating and pain relieving, the effect that relaxes bowel.Radix Angelicae Sinensis medicinal material main product is a kind of conventional Chinese medicine material in Minxian County, China Gansu, Wudu, Cheng County and other places, and resource is relatively nervous.And root and rhizome class medicinal materials such as Radix Angelicae Sinensis, genseng receive the infection of harmful germ in the course of cultivation especially easily, and the annual underproduction that causes because of Plant diseases reaches 10～30%, and the serious time, the outward appearance of medicinal material and interior quality descended simultaneously up to more than 50%.Organic pesticide is mainly adopted in the control of Chinese medicinal materials Plant diseases at present, and the result causes the Chinese medicinal materials pesticide residue to exceed standard, and has a strong impact on the quality and the security thereof of medicinal material, has become " bottleneck " that Chinese medicine goes to the world.In addition, in the cultivation of farm crop, damage to crops cultivated areas such as rice sheath blight disease, rice blast, bacterial leaf-blight, wheat powdery mildew, rust, head blight, nemas and virus disease reach 2,700 ten thousand hectares.Therefore, " nuisanceless " safety pesticide of efficient, the low toxicity of current urgent need development, low residue.And research and development have become current important research direction with the disease and pest that utilizes biological pesticide control Chinese medicinal materials and farm crop.Biological pesticide is meant and utilizes living microbe or its meta-bolites; Mainly be the one type of pesticide preparation that utilizes the meta-bolites of some peculiar microorganism or mikrobe that harmful organisms such as insect, germ, weeds, nematode, muroid are prevented and treated, or through bionical synthetic pesticide preparation with specific action.Have wide spectrum, efficient, safety, noresidue, no resistance and produce, do not murder natural enemy, advantage such as pollution of ecological environment not.For example prevent and treat white cuckoo disease and the genseng of the bighead atractylodes rhizome, chrysanthemum, the damping-off of Radix Panacis Quinquefolii with fungus Trichoderma; Utilize the black spot etc. of the new Polyoxin control of the meta-bolites of mikrobe genseng all to obtain good effect.
Endogenetic fungus is meant accomplishes the part or all of of its life history in plant materials; Grow between plant tissue cell; Be distributed in leaf sheath, seed, flower, stem, blade and the root, but the mikrobe that does not cause any illness has been highlighted the reciprocal symbiosis relation of endogenetic fungus and plant.In the microecosystem of endogenetic fungus and host plant formation, endogenetic fungus is being brought into play important ecological function, and 80% plant endogenesis epiphyte has stronger biological activity at aspects such as anti-algae or anti-weeds.Modern study shows that endogenetic fungus is the very abundant microbe groups of a variety, can produce some novel structures, the various secondary metabolite of type, like compounds such as terpene, alkaloids, aromatics, peptide classes.At present less about the research of medicinal plant endogenetic fungus anti-microbial activity; Radix Angelicae Sinensis research mainly concentrates on the research of activeconstituents and pharmacologically active aspect, and relevant report is not also seen in the research of Radix Angelicae Sinensis endogenetic fungus, therefore; On the basis of existing technology necessary; Make full use of the Radix Angelicae Sinensis resource, seek and to have the Radix Angelicae Sinensis endogenetic fungus and the metabolite thereof of anti-microbial activity, and the biological pesticide that is developed to the control plant pest has important application prospects.
Summary of the invention
Goal of the invention: the objective of the invention is in order to solve the deficiency of prior art; The Radix Angelicae Sinensis endogenetic fungus is furtherd investigate; A kind of active Radix Angelicae Sinensis endogenetic fungus of stronger anti-phytopathogen Fusella sp DG09 that has is provided, another object of the present invention provide fermented liquid of Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 and preparation method thereof with its as the application in the controlling plant diseases Agrotechnical formulation.
Technical scheme: in order to realize above purpose, the technical scheme that the present invention takes is:
A kind of have the active Radix Angelicae Sinensis endogenetic fungus of an anti-phytopathogen Fusella sp DG09; Fusella sp DG09 bacterial strain belongs to the mould genus of dark stalk shuttle spore Fusella sp fungi; Its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.4543; Preservation date: on January 17th, 2011, the preservation name is called: secretly obstruct the mould Fusella sp. of shuttle spore, the preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.Fusella sp DG09 strain characteristics is: the difference of conidium and mycelia is not obvious, and conidiophore and conidium are dead color, and conidium gives birth on the brachyplast of stalk, Dan Sheng, conidium fusiformis, no barrier film.
Separation method with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 provided by the invention is: gather fresh Radix Angelicae Sinensis explant, cut into segment after the water flushing, by follow procedure Radix Angelicae Sinensis explant surface is carried out disinfection then: using concentration earlier is 0.1% mercury chloride sterilization, 5～8min; Aseptic water washing 2～3 times; Using concentration again is 75% alcohol disinfecting 30 to 40 seconds, and aseptic water washing 2～3 times becomes the long segment of 0.3～0.5cm with the Radix Angelicae Sinensis explant re-shearing after the sterilization; Be inoculated on potato dextrose agar (PDA) substratum; Cultivated 8～10 days at 28 ℃, treat that Radix Angelicae Sinensis explant sample edge has mycelia to grow after, cut sample blocks edge mycelium inoculation and on the fresh PDA plate, continue cultivation; After waiting to grow fresh mycelia; Picking colony edge mycelium inoculation to the fresh PDA inclined-plane 28 ℃ cultivated 5～7 days, be forwarded to the fresh PDA inclined-plane so repeatedly 3～5 times, obtain Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09.Wherein said PDA substratum is processed by the raw material of following parts by weight: 20% yam (making method is: get yam and clean, be cut into small pieces, add an amount of zero(ppm) water, boil 20min after, filter, get filtrating), 2% glucose, 1.5～2.0% agar.
Fermented liquid with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 provided by the invention, it prepares through following method:
The slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 is inoculated into the shaking in the bottle of seed culture medium is housed, and in 28 ℃, the 200r/min shaking culture is to logarithmic phase; Cultured bacterial classification is inoculated into 500 liters of fermentor tanks that fermention medium is housed (volume ratio that is bacterial classification and fermention medium is 1: 10) by 10% inoculum size; Wherein the air flow of sterile air is 1: 0.6～1.2 (being the sterile air of feeding in the unit time and the volume ratio of fermention medium), and stirring velocity is 180～240 rev/mins, 25～30 ℃ of culture temperature; Incubation time 4～5 days; After the fermentation ends, centrifugal, get supernatant; Reduced vacuum is concentrated into 1/3～1/4 of original volume, gets Radix Angelicae Sinensis endogenetic fungus fermented liquid.
As preferred version, above-described fermented liquid with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09, wherein said seed culture medium is processed by the raw material of following parts by weight: 2% glucose; (making method is 0.3% groundnut meal: get groundnut meal, add an amount of zero(ppm) water, boiled 30 minutes; Filter, get filtrating), 0.3% sal epsom; 0.3% potassium primary phosphate, 0.3% ammonium chloride; Fermention medium is processed by the raw material of following parts by weight: 1% Zulkovsky starch (needing gelatinization), 1% glucose, 0.3% sal epsom, 0.3% potassium primary phosphate; 0.3% ammonium sulfate, (making method is 2% soybean cake powder: get soybean cake powder, add an amount of zero(ppm) water; Boiled 30 minutes, and filtered, get filtrating).
The Radix Angelicae Sinensis explant that the present invention adopted can be the positions such as root, stem or leaf of Radix Angelicae Sinensis.
Reactive site with fermented liquid of the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 provided by the invention, it prepares through following method:
The slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 is inoculated into the shaking in the bottle of seed culture medium is housed, and in 28 ℃, the 200r/min shaking culture is inoculated cultured bacterial classification into 500 liters of fermentor tanks that fermention medium is housed by 10% inoculum size to logarithmic phase; Wherein the air flow of sterile air is 1: 0.6～1.2, and stirring velocity is 180～240 rev/mins, 25～30 ℃ of culture temperature; Incubation time 4～5 days, after the fermentation ends, centrifugal; Get supernatant, reduced vacuum is concentrated into 1/3～1/4 of original volume, gets Radix Angelicae Sinensis endogenetic fungus fermented liquid; Earlier with petroleum ether extraction 1～3 time, and then with ethyl acetate extraction 1～3 time, combined ethyl acetate extraction liquid; Reduced vacuum concentrates, and drying obtains the reactive site of Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid.
Preparation of fermentation liquid method with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 provided by the invention, it may further comprise the steps:
The slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 is inoculated into the shaking in the bottle of seed culture medium is housed, and in 28 ℃, the 200r/min shaking culture is to logarithmic phase; Cultured bacterial classification is inoculated into 500 liters of fermentor tanks that fermention medium is housed by 10% inoculum size, and wherein the air flow of sterile air is 1: 0.6～1.2, and stirring velocity is 180～240 rev/mins; 25～30 ℃ of culture temperature, incubation time 4～5 days is after the fermentation ends; Centrifugal; Get supernatant, reduced vacuum concentrates, and gets the fermented liquid of Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09.
As preferred version, above-described have the active Radix Angelicae Sinensis endogenetic fungus of an anti-phytopathogen Fusella sp DG09 preparation of fermentation liquid method, and described seed culture medium is processed by the raw material of following parts by weight: 2% glucose; 0.3% groundnut meal (making method is: groundnut meal, add an amount of zero(ppm) water, and boiled 30 minutes; Filter, get filtrating), 0.3% sal epsom; 0.3% potassium primary phosphate, 0.3% ammonium chloride; Fermention medium is processed by the raw material of following parts by weight: 1% Zulkovsky starch (needing gelatinization), 1% glucose, 0.3% sal epsom, 0.3% potassium primary phosphate; 0.3% ammonium sulfate, (making method is 2% soybean cake powder: get soybean cake powder, add an amount of zero(ppm) water; Boiled 30 minutes, and filtered, get filtrating).
As preferred version, above-described have the active Radix Angelicae Sinensis endogenetic fungus of an anti-phytopathogen Fusella sp DG09 preparation of fermentation liquid method, and centrifugal speed is 5000 rev/mins, and centrifugation time is 10 to 15 minutes.
Fermented liquid or the reactive site with fermented liquid of the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 provided by the invention with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 provided by the invention can be used to prepare the controlling plant diseases Agrotechnical formulation.
As preferred version, the fermented liquid with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 provided by the invention or its efficient part are in the application in the disease Agrotechnical formulation such as mould, tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn early of preparation control gibberella saubinetii, tomato.
Beneficial effect: provided by the invention have the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 and fermented liquid thereof and compare with prior art and have the following advantages:
1, the present invention is through a large amount of experiment screenings; With fresh Radix Angelicae Sinensis plant is raw material; Filter out have anti-gibberella saubinetii, the tomato active useful endogenetic fungus Fusella sp DG09 of phytopathogen such as mould, tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn early; And the fermentating metabolism product of Fusella sp DG09 fungi carried out screening active ingredients, and filter out the strongest efficient part of anti-phytopathogen activity.The present invention separates fermented liquid or its efficient part obtain having the active endogenetic fungus Fusella of anti-phytopathogen sp DG09 from the Radix Angelicae Sinensis plant and is expected to become new biological pesticide resource; In the biological control of Plant diseases, will have application promise in clinical practice; And aspects such as the cultivation and production of Chinese medicinal materials and natural resources of Chinese medicinal materials sustainable use are all had important practical advice meaning be worth, and scientific basis is provided biological control and the development healthful Chinese crud drug of plant pathogenic fungi with theoretical.
2, the preparing method's environmental protection with anti-phytopathogen active Radix Angelicae Sinensis endogenetic fungus fermented liquid or its efficient part provided by the invention, its technological design is reasonable, and is workable, and the technological potentiality of product improvement are big, can realize suitability for industrialized production.
Embodiment
Further illustrate the present invention below in conjunction with specific embodiment; Should understand these embodiment only be used to the present invention is described and be not used in the restriction scope of the present invention; After having read the present invention, those skilled in the art all fall within the application's accompanying claims institute restricted portion to the modification of the various equivalent form of values of the present invention.
Embodiment 1
A kind of have the active Radix Angelicae Sinensis endogenetic fungus of an anti-phytopathogen Fusella sp DG09 fermented liquid, and it prepares through following method:
The slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 be inoculated into be equipped with seed culture medium (seed culture medium is processed by the raw material of following parts by weight: 2% glucose, 0.3% groundnut meal (is got groundnut meal, is added an amount of zero(ppm) water, boiled 30 minutes; Filter, get filtrating), 0.3% sal epsom, 0.3% potassium primary phosphate; Shaking in the bottle 0.3% ammonium chloride), in 28 ℃, the 200r/min shaking culture is to logarithmic phase, cultured bacterial classification inoculated by 10% inoculum size (fermention medium is processed by the raw material of following parts by weight: 1% Zulkovsky starch into fermention medium is housed; 1% glucose, 0.3% sal epsom, 0.3% potassium primary phosphate, 0.3% ammonium sulfate; 2% soybean cake powder (is got soybean cake powder, is added an amount of zero(ppm) water, boiled 30 minutes, filter; Get filtrating) 500 liters of fermentor tanks, wherein the air flow of sterile air is 1: 0.6～1, stirring velocity is 180～200 rev/mins, 25～30 ℃ of culture temperature; Incubation time 5 days, after the fermentation ends, centrifugal; Get supernatant, reduced vacuum is concentrated into 1/3 of original volume, gets Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid.
Embodiment 2
A kind of have the active Radix Angelicae Sinensis endogenetic fungus of an anti-phytopathogen Fusella sp DG09 fermented liquid, and it prepares through following method:
The slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 be inoculated into be equipped with seed culture medium (seed culture medium is processed by the raw material of following parts by weight: 2% glucose, 0.3% groundnut meal (is got groundnut meal, is added an amount of zero(ppm) water, boiled 30 minutes; Filter, get filtrating), 0.3% sal epsom, 0.3% potassium primary phosphate; Shaking in the bottle 0.3% ammonium chloride), in 28 ℃, the 200r/min shaking culture is to logarithmic phase, cultured bacterial classification inoculated by 10% inoculum size (fermention medium is processed by the raw material of following parts by weight: 1% Zulkovsky starch into fermention medium is housed; 1% glucose, 0.3% sal epsom, 0.3% potassium primary phosphate, 0.3% ammonium sulfate; 2% soybean cake powder (is got soybean cake powder, is added an amount of zero(ppm) water, boiled 30 minutes, filter; Get filtrating) 500 liters of fermentor tanks, wherein the air flow of sterile air is 1: 0.8～1.2, stirring velocity is 200～240 rev/mins, 25～30 ℃ of culture temperature; Incubation time 4 days, after the fermentation ends, centrifugal; Get supernatant, reduced vacuum is concentrated into 1/4 of original volume, gets Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid.
Embodiment 3
A kind of reactive site with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 fermented liquid, it prepares through following method:
The slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 be inoculated into be equipped with seed culture medium (the seed culture based formulas is: 2% glucose, 0.3% groundnut meal (is got groundnut meal, is added an amount of zero(ppm) water, boiled 30 minutes, filter; Get filtrating), 0.3% sal epsom, 0.3% potassium primary phosphate, 0.3% ammonium chloride) shake in the bottle; In 28 ℃, the 200r/min shaking culture is to logarithmic phase, cultured bacterial classification inoculated by 10% inoculum size (fermentative medium formula is: 1% Zulkovsky starch, 1% glucose into fermention medium is housed; 0.3% sal epsom, 0.3% potassium primary phosphate, 0.3% ammonium sulfate, 2% soybean cake powder (is got soybean cake powder; Add an amount of zero(ppm) water, boiled 30 minutes, filter, get filtrating) 500 liters of fermentor tanks; Wherein the air flow of sterile air is 1: 0.8～1.2, and stirring velocity is 180～200 rev/mins, 28～30 ℃ of culture temperature, incubation time 5 days; After the fermentation ends, centrifugal, get supernatant, reduced vacuum is concentrated into 1/3 of original volume; Radix Angelicae Sinensis endogenetic fungus fermented liquid, earlier with petroleum ether extraction 3 times, and then, extract respectively 3 times with 50% ethanol and water more successively then with ethyl acetate extraction 3 times; Merge above acetic acid ethyl acetate extract, reduced vacuum concentrates, and drying promptly obtains the lower ETHYLE ACETATE reactive site of Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid Semi-polarity.
Embodiment 4
A kind of reactive site with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 fermented liquid, it prepares through following method:
The slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 be inoculated into be equipped with seed culture medium (the seed culture based formulas is: 2% glucose, 0.3% groundnut meal (is got groundnut meal, is added an amount of zero(ppm) water, boiled 30 minutes, filter; Get filtrating), 0.3% sal epsom, 0.3% potassium primary phosphate, 0.3% ammonium chloride) shake in the bottle; In 28 ℃, the 200r/min shaking culture is to logarithmic phase, cultured bacterial classification inoculated by 10% inoculum size (fermentative medium formula is: 1% Zulkovsky starch, 1% glucose into fermention medium is housed; 0.3% sal epsom, 0.3% potassium primary phosphate, 0.3% ammonium sulfate, 2% soybean cake powder (is got soybean cake powder; Add an amount of zero(ppm) water, boiled 30 minutes, filter, get filtrating) 500 liters of fermentor tanks; Wherein the air flow of sterile air is 1: 0.6～1.0, and stirring velocity is 200～240 rev/mins, 28～30 ℃ of culture temperature, incubation time 4 days; After the fermentation ends, centrifugal, get supernatant, reduced vacuum is concentrated into 1/4 of original volume; Radix Angelicae Sinensis endogenetic fungus fermented liquid, earlier with petroleum ether extraction 3 times, and then, extract respectively 3 times with 50% ethanol and water more successively then with ethyl acetate extraction 3 times; Merge above acetic acid ethyl acetate extract, reduced vacuum concentrates, and drying promptly obtains the lower ETHYLE ACETATE reactive site of Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid Semi-polarity.
The determination of activity of embodiment 5 anti-plant pathogenic fungi mycelial growths
1, antagonistic experiment: the picking diameter is that the plant pathogenic fungi bacterium piece of 6mm is inoculated in the dull and stereotyped central authorities of PDA; To from the Radix Angelicae Sinensis explant, separate the endogenetic fungus Fusella sp DG09 bacterium piece (diameter: 6mm) that obtains then; Be seeded in around the PDA flat board, cultivated 6 days in 28 ℃.
2, suppress the mycelial growth method: with the PDA substratum mixing of Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid 1ml, pour into and process the culture medium flat plate that contains the endogenetic fungus fermented liquid in the sterile petri dish with the 9ml thawing, treat culture medium solidifying after; Put into 1 bacterium cake (diameter is 6mm) that supplies the examination bacterium; Make the one side that the bacterium cake contains mycelia be attached to media surface, handle repetition 5 times for every kind, behind 28 ℃ of cultivation 72～96h; Measure colony diameter, be the treatment group colony diameter; Set up control group simultaneously, promptly do not containing the corresponding plant pathogenic fungi of inoculation on the PDA culture medium flat plate of endogenetic fungus fermented liquid, other condition is identical with treatment group.Calculate inhibiting rate by following formula.
Mycelial growth inhibition rate (%)=(control group colony diameter-treatment group colony diameter)/(control group colony diameter-6) * 100%
3, result and analysis
3.1 Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 is to the restraining effect of plant pathogenic fungi mycelial growth
With two parts of the fermented liquids (embodiment 1 and 2) of the endogenetic fungus Fusella sp DG09 (preserving number is CGMCC No.4543) that is located away from the fresh explant of Radix Angelicae Sinensis; Carry out respectively to gibberella saubinetii, tomato early mould, the tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn antagonistic experiment and suppress the mycelial growth experiment; Experimental result shows; Radix Angelicae Sinensis endogenetic fungus Fusella spDG09 antagonistic action provided by the invention is more general; Early mould, the tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn all have stronger antagonistic action to gibberella saubinetii, tomato; And experimental result shows Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid, and early mould, the tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn all have stronger restraining effect to gibberella saubinetii, tomato; Especially the restraining effect to gibberella saubinetii and tomato gray mould growth is remarkable, and inhibiting rate reaches 42.5% and 76.6% respectively, and concrete experimental result is as shown in table 1.
Table 1 Radix Angelicae Sinensis endogenetic fungus Fusella spDG09 is to the restraining effect of plant pathogenic fungi mycelial growth
3.2 the screening at Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid bacteriostatic activity position
Sherwood oil, ETHYLE ACETATE, 50% ethanol, the water opposed polarity solvent extractable matter of the Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid that will prepare by the embodiment of the invention 3 carry out bacteriostatic activity and detect.Concrete experimental result is as shown in table 2; Show by experimental result; The ethyl acetate extraction position is stronger to the mycelial restraining effect of plant pathogenic fungi in the Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid, and it is more to be illustrated in activity substance content nonpolar in the endogenetic fungus secondary metabolite, close ester property.
The different extract parts of table 2 Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid are to the restraining effect of pathogenic fungi mycelial growth
The determination of activity of embodiment 6 anti-plant pathogenic fungi spore germinations
1, suppress pathogenic fungi spore germination experiment: get gibberella saubinetii, tomato early the spore of test plant pathogenic fungi such as mould, tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn be made into the spore suspension of proper concn; Observe at 10 * 10 low power lens, there are 30～40 spores in each visual field.1ml Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid is mixed with the 1ml spore suspension, get 1 dropping then on depression slide, handle repetition 5 times for every kind.At 25 ℃; 80～90% humidity condition is cultivated down; Is sprouting with spore germ tube length greater than spore short radius person, after the spore germination rate of control group fungi reaches 80%, calculates the germination rate of all Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermentation liquor treatment groups.Spore count * 100% that spore germination inhibiting rate (%)=(spore count of spore count-treatment group sprouting that control group is sprouted)/control group is sprouted
2, result and analysis
2.1 Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 is to the restraining effect of plant pathogenic fungi spore germination
The fermented liquid of the endogenetic fungus Fusella sp DG09 that is located away from the fresh explant of Radix Angelicae Sinensis (embodiment 1 and embodiment 2) is carried out respectively the spore germination of mould, the tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn early of gibberella saubinetii, tomato is suppressed experiment; Experimental result shows; The spore germination of mould, the tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn early all has stronger restraining effect to Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid provided by the invention to gibberella saubinetii, tomato; Inhibiting rate reaches about 90%, and concrete experimental result is as shown in table 3.
Table 3 Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid is to the restraining effect of plant pathogenic fungi spore germination
2.2 Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid suppresses the reactive site screening of plant pathogenic fungi spore germination
Sherwood oil, ETHYLE ACETATE, 50% ethanol, the water opposed polarity solvent extractable matter of the Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid that will prepare by the embodiment of the invention 3, the activity that suppresses fungus spore germination detects.Concrete experimental result is as shown in table 4; Experimental result by table 4 shows; The ethyl acetate extraction position is stronger to the spore germination restraining effect of plant pathogenic fungi in the Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid, and it is more to be illustrated in the endogenetic fungus Fusella sp DG09 secondary metabolite activity substance content nonpolar, close ester property.
The different extract parts of table 4 Radix Angelicae Sinensis endogenetic fungus Fusella spDG09 fermented liquid are to the restraining effect of plant pathogenic fungi spore germination
Show through above experimental result; The present invention screens the Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 that obtains from the Radix Angelicae Sinensis plant; And Radix Angelicae Sinensis endogenetic fungus Fusella spDG09 carried out the reactive site that fermentation culture obtains Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 fermented liquid or Radix Angelicae Sinensis endogenetic fungus Fusella sp fermented liquid have the activity of stronger inhibition phytopathogen, as to gibberella saubinetii, tomato early germ such as mould, tomato gray mould of epidemic disease and Rhizoctonia solani Kuhn all have stronger restraining effect.The present invention makes full use of the Radix Angelicae Sinensis medicine resource; Separate to obtain having the active endogenetic fungus Fusella of anti-phytopathogen sp DG09 and fermented liquid or its efficient part thereof and be expected to develop and become new biological pesticide resource, in the biological control of plant pest, have application promise in clinical practice.
The above only is a preferred implementation of the present invention; Should be pointed out that for those skilled in the art, under the prerequisite that does not break away from the principle of the invention; Can also make some improvement and retouching, these improvement and retouching also should be regarded as protection scope of the present invention.
Claims (10)
1. one kind has the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09; It is characterized in that; Fusella sp DG09 bacterial strain belongs to the mould genus of dark stalk shuttle spore Fusella sp fungi; Its preserving number at China Committee for Culture Collection of Microorganisms common micro-organisms center is CGMCC No.4543, preservation date: on January 17th, 2011.
2. fermented liquid with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 is characterized in that it prepares through following method: the slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 is inoculated into the shaking in the bottle of seed culture medium is housed; In 28 ℃, the 200r/min shaking culture is inoculated cultured bacterial classification into 500 liters of fermentor tanks that fermention medium is housed by 10% inoculum size to logarithmic phase; Wherein the air flow of sterile air is 1: 0.6～1.2, and stirring velocity is 180～240 rev/mins, 25～30 ℃ of culture temperature; Incubation time 4～5 days; After the fermentation ends, centrifugal, get supernatant; Reduced vacuum is concentrated into 1/3～1/4 of original volume, gets Radix Angelicae Sinensis endogenetic fungus fermented liquid.
3. the fermented liquid with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 according to claim 2; It is characterized in that; Described seed culture medium is processed by the raw material of following parts by weight: 2% glucose, 0.3% groundnut meal, 0.3% sal epsom; 0.3% potassium primary phosphate, 0.3% ammonium chloride; Described fermention medium is processed by the raw material of following parts by weight: 1% Zulkovsky starch, 1% glucose, 0.3% sal epsom, 0.3% potassium primary phosphate, 0.3% ammonium sulfate, 2% soybean cake powder.
4. active part with fermented liquid of the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09, it is characterized in that it prepares through following method: the weighting profit requires the fermented liquid of the 2 prepared Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 that obtain; Use earlier petroleum ether extraction; And then use ethyl acetate extraction, and the combined ethyl acetate extraction liquid, reduced vacuum concentrates; Drying obtains the active part of Radix Angelicae Sinensis endogenetic fungus fermented liquid.
5. the described preparation of fermentation liquid method with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 of claim 2 is characterized in that, may further comprise the steps:
The slant strains of getting Radix Angelicae Sinensis endogenetic fungus Fusella sp DG09 is inoculated into the shaking in the bottle of seed culture medium is housed, and in 28 ℃, the 200r/min shaking culture is to logarithmic phase; Cultured bacterial classification is inoculated into 500 liters of fermentor tanks that fermention medium is housed by 10% inoculum size, and wherein the air flow of sterile air is 1: 0.6～1.2, and stirring velocity is 180～240 rev/mins; 25～30 ℃ of culture temperature, incubation time 4～5 days is after the fermentation ends; Centrifugal; Get supernatant, reduced vacuum is concentrated into 1/3～1/4 of original volume, gets Radix Angelicae Sinensis endogenetic fungus fermented liquid.
6. the preparation of fermentation liquid method with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 according to claim 5; It is characterized in that; Described seed culture medium is processed by the raw material of following parts by weight: 2% glucose, 0.3% groundnut meal, 0.3% sal epsom; 0.3% potassium primary phosphate, 0.3% ammonium chloride; Fermention medium is processed by the raw material of following parts by weight: 1% Zulkovsky starch, 1% glucose, 0.3% sal epsom, 0.3% potassium primary phosphate, 0.3% ammonium sulfate, 2% soybean cake powder.
7. claim 2 or the 3 described application of fermented liquid in preparation controlling plant diseases Agrotechnical formulation with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09.
8. the application of fermented liquid in preparation controlling plant diseases Agrotechnical formulation with the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 according to claim 7; It is characterized in that described Plant diseases comprises gibberella saubinetii, tomato early mould, tomato gray mould of epidemic disease and rice sheath blight disease fungus diseases.
9. the described application of active part in preparation controlling plant diseases Agrotechnical formulation of claim 4 with fermented liquid of the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09.
10. the application of active part in preparation controlling plant diseases Agrotechnical formulation with fermented liquid of the active Radix Angelicae Sinensis endogenetic fungus of anti-phytopathogen Fusella sp DG09 according to claim 9; It is characterized in that described Plant diseases comprises gibberella saubinetii, tomato early mould, tomato gray mould of epidemic disease and rice sheath blight disease fungus diseases.
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| CN108040752B (en) * | 2017-12-01 | 2020-03-31 | 南京中医药大学 | Artificial cultivation method of isaria cicadae and traditional Chinese medicine cordyceps sobolifera |
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| CN101037656A (en) * | 2007-01-25 | 2007-09-19 | 中国计量学院 | Horned holly fungal endophyte-harzianum L |
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| CN101037656A (en) * | 2007-01-25 | 2007-09-19 | 中国计量学院 | Horned holly fungal endophyte-harzianum L |
| CN101041840A (en) * | 2007-01-25 | 2007-09-26 | 中国计量学院 | Preparation method of sesquiterpenoids Trichothec-9-en-4-o1,12,13-epoxy-,acetate,(4beta)-(8CI,9CI) |
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