CN105462847B - Application of the sophora tonkinensis Gapnep endogenetic fungus TRXY-46 in prevention and treatment notoginseng root rot - Google Patents

Abstract

The invention discloses a kind of sophora tonkinensis Gapnep endogenetic fungus TRXY-46, the classification naming of sophora tonkinensis Gapnep endogenetic fungus TRXY-46 is Rhexocercosporidium sp.TRXY-46, the ITS sequence of bacterial strain is as described in SEQ ID NO.1, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of Microorganism, Academia Sinica, preservation date: on December 03rd, 2014, deposit number: CGMCC No.10108.The present invention for the first time from the root of medicinal plant sophora tonkinensis Gapnep separation screening to one plant of endogenetic fungus (Rhexocercosporidium sp.) TRXY-46, the bacterium has very strong inhibiting effect to notoginseng root rot bacterium, brings wide application prospect for the field of biological control of Radix Notoginseng fungal disease.

Description

Application of the sophora tonkinensis Gapnep endogenetic fungus TRXY-46 in prevention and treatment notoginseng root rot

Technical field

The present invention relates to field of biotechnology, in particular to a kind of sophora tonkinensis Gapnep endogenetic fungus TRXY-46 is in prevention and treatment Roots of Panax Notoginseng Application in maize ear rot.

Background technique

Modern agriculture is overly dependent upon the use of chemical pesticide to the prevention and treatment of plant disease, and the release of a large amount of chemical pesticides is not Only there is long-term destruction to ecological environment, also quality of agricultural product is caused to decline, excessive pesticide residues, the drug resistance of pathogen Property and to problems such as people and animals' nocuousness.It finds safer, effective pest control method to be of great significance, utilize The method of biology, which carrys out controlling plant diseases, effectively to solve the above problems.

Radix Notoginseng (Panax notoginseng F.H.chen) also known as pseudo-ginseng, invaluable etc., have significant activating microcirculation and removing stasis medicinal, Detumescence ding-tong effect is a kind of Chinese tradition rare medicinal herbs.Using Radix Notoginseng as Chinese patent drug made of primary raw material such as " Yunnan Baiyao " " Pien Tze Huang " etc. is wide-spread.In recent years, the demand to Radix Notoginseng raw material is growing.But the nosomycosis in cultivation Evil has seriously affected Panax notoginseng Growth.Currently, prevention and treatment fungal disease depends on chemical pesticide unduly on notoginseng planting, a large amount of chemistry The use of pesticide not only affects the quality of Radix Notoginseng, also results in a large amount of pesticide residues of pseudo-ginseng.

Notoginseng root rot is one of Radix Notoginseng Major Diseases, is commonly called as " green smelly " in producing region, cardinal symptom be seedling stage bud-rot and The water stain shape lesion of brown of its reed head and bastem junction is until spread to entire stem base portion.Just there is generation in the disease seeding stage, 4, disease in May is stagnated, and the 6-9 month enters rainy season, into onset peak period.There are many types for root rot, it was reported that its symptom Mostly caused by notoginseng root rot bacterium (Fusarium solani) (abbreviation F.solani).

This fungal disease is one of the main reason for causing Radix Notoginseng product quality for many years and yield to decline.In Guangxi three Seven traditional Genuine producing area, due to low altitude area and the meteorological condition of high temperature and humidity, this disease is often simultaneous, causes The a large amount of underproduction of Radix Notoginseng are even had no harvest, and are had brought tremendous economic losses to farmer.In order to control this fungal disease, greatly The chemical pesticide of amount is used, and a large amount of pesticide residues of Radix Notoginseng product are caused, serious to polluted environment, is greatly threatened People's health.Therefore, development biological control Radix Notoginseng fungal disease is very urgent to the sustainable development of Radix Notoginseng industry and must It wants.The Antagonistic Fungi for filtering out energy while this disease of antagonism thus is of great significance, and screens and then develops and uses Antagonistic Fungi It is also effectively to control one of most potential control measure of Radix Notoginseng fungal disease.

The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.

Summary of the invention

The purpose of the present invention is to provide a kind of sophora tonkinensis Gapnep endogenetic fungus TRXY-46 answering in prevention and treatment notoginseng root rot With so as to the notoginseng root rot occurred during effectively preventing notoginseng planting, to improve the yield and quality of Radix Notoginseng.

To achieve the above object, technical solution provided by the invention is as follows:

The classification naming of a kind of sophora tonkinensis Gapnep endogenetic fungus TRXY-46, sophora tonkinensis Gapnep endogenetic fungus TRXY-46 is Rhexocercosporidium sp.TRXY-46, the ITS sequence of bacterial strain is as described in SEQ ID NO.1, depositary institution: China is micro- Biological inoculum preservation administration committee common micro-organisms center, preservation address: in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Institute of microbiology of the academy of sciences of state, preservation date: on December 03rd, 2014, deposit number: CGMCC No.10108.

Preferably, the sophora tonkinensis Gapnep endogenetic fungus TRXY-46 metabolite the preparation method comprises the following steps:

(1) sophora tonkinensis Gapnep endogenetic fungus TRXY-46 is inoculated in potato dextrose agar plane ware (PDA plate) In, being placed in temperature is to cultivate 20 days at 28 DEG C, and gained culture materials are cut into bulk and are transferred in sterile solid culture medium, are placed in 28 DEG C ferment 60 days；

(2) after fermentation, the methanol soaking fermentation object and ultrasound 40min with 2 times of fermentation material, is then filtered；

(3) step (2) are repeated twice, merges filtrate twice and medicinal extract is concentrated under reduced pressure into, obtains endogenetic fungus TRXY-46 The methanol crude extract of fermentation material, as sophora tonkinensis Gapnep endogenetic fungus TRXY-46 metabolite.

Preferably, sterile solid culture medium potato containing 400g described in step (1), the dextrose and 20g sugarcane of 20g Sugar.

The answering in prevention and treatment notoginseng root rot of the metabolite of sophora tonkinensis Gapnep endogenetic fungus TRXY-46 as obtained by above-mentioned preparation With.

Compared with prior art, the invention has the following beneficial effects:

The present invention for the first time from the root of medicinal plant sophora tonkinensis Gapnep separation screening to one plant of endogenetic fungus (Rhexocercosporidium sp.) TRXY-46, the bacterium have very strong inhibiting effect to notoginseng root rot bacterium, are Radix Notoginseng The field of biological control of fungal disease brings wide application prospect.

Detailed description of the invention

Fig. 1 is the plate opposite culture of sophora tonkinensis Gapnep endogenetic fungus TRXY-46 and notoginseng root rot of the present invention, and wherein F is three Seven pine root fungus (Fusarium solani), a are blank control, and b is experimental group.

Fig. 2 is sophora tonkinensis Gapnep endogenetic fungus TRXY-46 strain morphology feature of the present invention.

Fig. 3 is systematic evolution tree of the sophora tonkinensis Gapnep endogenetic fungus TRXY-46 bacterial strain based on ITS sequence.

Fig. 4 is bacterium of the metabolite to notoginseng root rot bacterium of bacterial strain sophora tonkinensis Gapnep endogenetic fungus TRXY-46 according to the present invention The inhibitory effect of silk growth；Wherein the 1st, the 5th for blank control be not drug containing PDA plate；2nd, the 3rd, the 4th is the positive Compare powder of carbendazim；6th, the 7th, the 8th is the metabolite of bacterial strain, and concentration is respectively 2mg/ml, 4mg/ml, 8mg/ml；F For notoginseng root rot bacterium Fusarium solani.

Specific embodiment

It is described in detail With reference to embodiment, it is to be understood that protection scope of the present invention is not by specific The limitation of embodiment.Experimental method used in following embodiments is conventional method unless otherwise specified.Following implementations Material, reagent used in example etc., is commercially available unless otherwise specified.Methanol is the commercially available pure methanol of analysis. 2X TagMasterMix is purchased from precious bioengineering Co., Ltd (Takara), and Primer-1, Primer-2 are closed by Hua Da gene At.

Embodiment 1

Separation, the screening and identification of bacterial strain

One, the separation of bacterial strain

Material to be tested: the wild sophora tonkinensis Gapnep of Guangxi Tiandeng County In Limestone Area is picked up from.

Strains tested: it is provided by plant pathology research institute of Guangxi University.

Culture medium: 1000ml potato dextrose agar (PDA culture medium), PDA culture medium 300g containing potato, Glucose 20g, agar 20g and chloramphenicol 0.1g, Medium's PH Value are 6.0 ± 0.2.

Surface sterilization: a length of 6-8cm, width is dry to rush for the sophora tonkinensis Gapnep root flowing water flushing 30min of 1-2cm fresh and healthy Net silt air-dries surface moisture, superclean bench is moved on to, sterile then by clean sophora tonkinensis Gapnep root with rinsed with sterile water 2 times Under the conditions of, by sophora tonkinensis Gapnep root volumetric concentration be 75% ethyl alcohol impregnate 1min, rinsed with sterile water 2 times, sodium hypochlorite (effective chlorine 1%) 2min is impregnated, sterile washing 3 times, it is spare that sterile blotting paper blots surface.

The separation of bacterial strain purifies: the good sophora tonkinensis Gapnep root of surface sterilization, under aseptic condition, scrapes off epidermis with sterile wood chip, uses Sterile secateurs cuts off the both ends of sophora tonkinensis Gapnep root, and the sterile pocket knife of remaining part and aseptic nipper separate xylem and bast, and The tissue block of 0.5cm long is cut into sterile secateurs, it is sterile to be forwarded to the PDA culture medium (containing chloramphenicol) prepared, 28 DEG C of cultures. Surface sterilization last time rinsing liquid is taken to be coated on PDA plate, as negative control.Observation daily, wait be grown around organizing Mycelia, the single mycelia of picking, is connected to the PDA culture medium of antibiotic-free.The bacterium colony grown continues if colonial morphology is inconsistent The single mycelia switching PDA culture medium of picking, until the formalness of the bacterium colony newly grown on plate is consistent.

The endogenetic fungus being separated to is inoculated in PDA plate, it is stand-by after being cultivated 20 days at 28 DEG C.By notoginseng root rot bacterium It is inoculated in PDA plate, it is stand-by after being cultivated 3 days at 28 DEG C.

Two, it screens

Using tablet face-off method to the primary dcreening operation for carrying out thallus bacteriostatic activity for examination sophora tonkinensis Gapnep endogenetic fungus.

Firstly, 6mm bacteria cake is made in endogenetic fungus and notoginseng root rot bacterium with sterilization punchers under aseptic condition；Locating In reason group, endogenetic fungus bacteria cake is transferred to the center of the culture dish containing PDA, then the isometrical 3cm around the bacteria cake of endogenetic fungus Notoginseng root rot bacterium bacteria cake is placed at place, and every plant of endogenetic fungus repeats 3 wares；In control group, the culture dish center of PDA does not connect interior life Fungi bacteria cake places notoginseng root rot bacterium bacteria cake at the culture dish center isometrical 3cm of surrounding of PDA, repeats 3 wares.Then 28 DEG C Lower culture, periodic observation.Radix Notoginseng root-rot is measured in processing group to the long culture dish plate center to PDA of mycelia in control group The growth radius of germ bacteria cake center to notoginseng root rot bacterium between endogenetic fungus bacteria cake center is processing growth radius；It is compareing In group, the growth radius at measurement notoginseng root rot bacterium bacteria cake center to notoginseng root rot bacterium between the culture dish center of PDA is pair According to growth radius.Finally, according to the following formula, calculating bacteriostasis rate:

It compares increment=control and grows radius-bacteria cake radius

It handles increment=processing and grows radius-bacteria cake radius

As a result 4 plants are obtained to all very strong antagonism sophora tonkinensis Gapnep endogenetic fungal bacterial strain of notoginseng root rot bacterium inhibiting effect, In one plant of entitled TRXY-46, be 58% to the inhibiting rate of notoginseng root rot bacterium.

Three, it identifies

(1) strain morphology feature

Morphological features observation: endophyte fungal bacterial strain TRXY-46 to be identified is seeded in PDA culture medium, is set It is cultivated in 28 DEG C, observed its cultural characteristic and color change at 5,14 and 20 days respectively.Take the color characteristic conduct for stablizing maturation Its cultural characteristic, the foundation as identification.Observe and record the color of aerial hyphae, the size of bacterium colony, color, tissue profile, table Face shape etc. is used as fixed reference feature.

Spore shape observation of characteristics: endophyte fungal bacterial strain TRXY-46 to be identified is made into inserted sheet culture, is trained when in PDA Support base on do not produce spore, by mycelia switching on the low nutrition culture medium (a quarter concentration PDA) containing sterile sophora tonkinensis Gapnep root with Inducing spore, by observed form result microphotograph, as shown in Figure 2.

(2) bacterial strain ITS sequence and its phylogenetic analysis

The preparation of DNA profiling:

Reagent: (1) lysis buffer: 1% (w/ of Tris-Ac (pH 7.8) 40mM, NaAc 20mM, EDTA 1mM, SDS v)；

(2) 5M NaCl solution.

DNA is extracted:

Extracting solution (Tris-Ac (pH 7.8) 40mM, the NaAc 20mM, EDTA1mM, SDS for adding 600 μ L65 DEG C to preheat 1%) 1.5ml centrifuge tube (EP pipe) is arrived, scrapes a small amount of mycelia with stick and is put into the grinding of EP pipe, 65 DEG C of water-bath 30min, centrifugal rotational speed 12000r/min is centrifuged 10min；

Centrifugation gained 400 μ L of supernatant is taken, 100 μ L of 5M NaCL, ice bath 10min are added；

Then it is 12000r/min that centrifugal rotational speed is kept at being 4 DEG C in temperature, is centrifuged 10min, takes supernatant；In addition clear liquid 0.6 times of isopropanol and supernatant mix (or 2 times of dehydrated alcohols) ice bath 1h, keep centrifugal rotational speed 12000r/min, centrifugation 10min, the remaining substance of gained dries after discarding supernatant liquid, adds 20 μ L distilled water (ddH₂O), 1-2 μ L is taken to do DNA profiling.

PCR amplification ITS sequence:

(1) PCR instrument: ABI 3730-XL DNA sequencer (Applied Biosystems, USA)

(2) amplimer: ITS1 (5 '-TCCGTAGGTGAACCTGCGG-3 ') is as shown in SEQ ID NO.2 and ITS4 (5 '-TCCTCCGCTTATTGATATGC-3 ') are as shown in SEQ ID NO.3；

(3) amplification system:

ITS sequence PCR amplification system is as shown in table 1: table 1.

Reactant	Sample-adding amount
		2X TagMasterMix	25μL
Primer-1	1μL
		Primer-2	1μL
Template DNA	2μL
		ddH2O	21μL
React total volume	50μL

Note: DNA profiling is made to be above-mentioned in the Template DNA in table 1.

PCR reaction condition is as shown in table 2:

Table 2.

Note: step 2 carries out 30 circulations in table 2.

The electrophoresis detection of pcr amplification product:

The Ago-Gel (the 5 μ l/100ml containing Goldview) that deposition condition is 1%, 1 × TBE electrophoretic buffer, 90V Electrophoresis 1 hour, PCR product applied sample amount was 3 μ L, point sample after mixing with 1 μ L Loading dye.In the ultraviolet lower sight of 254nm It examines as a result, determining expanding fragment length using the DL1000DNA Marker of TaKaRa company as nucleic acid standard molecular weight object of reference. Amplified production band should be on the position of reference substance 400-700bp.

PCR product purifying and sequencing: it is carried out by Shenzhen Huada Genetic Technology Co., Ltd.

The building of systematic evolution tree:

The ITS sequence of the fungi measured in the ITS sequence surveyed and GenBank gene pool is compared, according to than Correlated series are downloaded to result.Network analysis is carried out simultaneously by the adjoining algorithm (Neighbor-joining NJ) of MEGA 6.0 Systematic evolution tree is constructed, as shown in Figure 3.

As a result:

(1) strain morphology feature

Bacterium colony size: it is better than being grown in the PDA culture medium of commercialization in artificial PDA culture medium, on artificial PDA, bacterium Limitation growth is fallen, slow growth is cultivated one month, bacterium colony fusiform, diameter 16mm, height 3mm；

Colony colour: bacterium colony is just flushing green, and reverse side is pink, and edge is greyish white, and culture medium is slightly red；

The tissue profile of bacterium colony: mycelia forms close villiform, and colony edge is wavy；

The surface shape of bacterium colony: there is water droplet on close mycelia, bacterium colony surface；

Mycelia: mainly aerial hyphae, no spore, old mycelia is red, and new mycelia is grayish green.

Spore shape feature: through inducing, no spore.

(2) bacterial strain ITS sequence and its phylogenetic analysis

Using primer I TS1 and ITS4, the segment of a 500-600bp size is amplified from strain gene group DNA, is passed through It is sequenced and compares sequencing result by BLASTn in GenBank, relevant reference strain sequence, benefit are downloaded according to comparison result Network analysis is carried out with the adjoining algorithm (Neighbor-joining NJ) of MEGA6 and constructs systematic evolution tree.The result shows that It is 93% that bacterial strain TRXY-46 and Rhexocercosporidium sp.Dzf14 (EU543257), which get together and to form holding strength, End branch.Base similarity system design the result shows that, TRXY-46 and Rhexocercosporidium sp.Dzf14 (EU543257) 3 bases, sequence similarity 99.3% are differed.Comprehensive morphological and molecular biological characteristics, will be at the beginning of bacterial strain Step is accredited as Rhexocercosporidium sp..

Embodiment 2

Inhibiting effect of the metabolite of bacterial strain to notoginseng root rot bacterium

One, the extraction of the fermented and cultured of bacterium and tunning

(1) sophora tonkinensis Gapnep endogenetic fungus TRXY-46 is inoculated in potato dextrose agar plane ware (PDA plate) In, being placed in temperature is to cultivate 20 days at 28 DEG C, and gained culture materials are cut into bulk and are transferred to sterile solid culture medium (containing 400g Potato, the dextrose of 20g and 20g sucrose) 2 liters of conical flask in, ferment 60 days under the conditions of being placed in 28 DEG C；

(2) after fermentation, the methanol soaking fermentation object and ultrasound 40min with 2 times of fermentation material, then uses filtered through gauze, Take filtrate；

(3) step (2) are repeated twice, merges filtrate twice and medicinal extract is concentrated under reduced pressure into, obtains endogenetic fungus TRXY-46 The methanol crude extract of fermentation material, as sophora tonkinensis Gapnep endogenetic fungus TRXY-46 metabolite.

Two, inhibiting effect of the sophora tonkinensis Gapnep endogenetic fungus TRXY-46 metabolite to the mycelia growth of notoginseng root rot bacterium

The inhibition grown with the metabolite of mycelia growth method measurement bacterial strain TRXY-46 to notoginseng root rot bacterium mycelia is living Property.The metabolite of bacterial strain TRXY-46 and powder of carbendazim (positive control) are respectively prepared containing 2mg/mL, 4mg/mL, 8mg/ The drug containing tablet of mL concentration metabolite.Aseptically, 6mm bacteria cake is made in notoginseng root rot bacterium with punch, three Seven pine root fungus bacteria cakes are connected to each drug containing tablet center as processing, and the notoginseng root rot bacterium bacteria cake connect with not drug containing tablet center is Negative control, in triplicate, all plates are placed in 28 DEG C of cultures for all processing and control.Drug concentration presses the field of powder of carbendazim Between using concentration be arranged.When negative control covers with culture dish, measurement compares bacterium colony and handles the growth diameter of bacterium colony respectively, and According to the following formula, inhibiting rate is calculated:

Negative control increment=negative control growth diameter-bacteria cake diameter

Handle increment=processing growth diameter-bacteria cake diameter

Three, minimal inhibitory concentration of the sophora tonkinensis Gapnep endogenetic fungus TRXY-46 metabolite to notoginseng root rot bacterium

With the metabolite of broth dilution method determination bacterial strain TRXY-46 to the minimal inhibitory concentration of notoginseng root rot bacterium.It will 5 days notoginseng root rot bacterium pure culture biscuits involvng inoculations are cultivated in PDA culture medium in the PDB fluid nutrient medium for containing 0.2% Tween 80 (v/v) In, 28 DEG C, 150r/min shaking table culture 7 days, culture is then gone into triangular flask, and pour into containing the sterile of 0.2% Tween 80 Distilled water stirs 30min with magnetism stick, solution is filtered with sterile gauze, obtains spores solution, and with blood counting chamber by spore Sub- concentration is adjusted to every milliliter 10⁴A spore is spare.The metabolite of bacterial strain TRXY-46 is dissolved into 80mg/mL with 1%DMSO Metabolite concentration for the treatment of, take 5 sterile test tubes to be sequentially arranged on rack for test tube, number is 1,2,3,4,5 respectively, every pipe point Not Jia Ru 1ml contain the aseptic double-distilled water of 0.2% Tween 80, then the bacterial strain methanol crude extract solution of 1ml 80mg/mL is added In the test tube that number is 1 and successively carry out twice of serial dilution in each pipe, and by the above-mentioned gained spores solution (10 of 1ml⁴A/ Ml it) is separately added into each pipe, to obtain the metabolite concentration for the treatment of of final TRXY-46: 20mg/ml (1 test tube of number), (number 5 is tried by 10mg/ml (2 test tube of number), 5mg/ml (3 test tube of number), 2.5mg/ml (4 test tube of number), 1.25mg/ml Pipe).The 8mg/mL Flusilazole of the 1%DMSO and 1ml of 1ml replace metabolite to execute above-mentioned treatment process and conduct respectively Negative control and positive control, all processing and control are in triplicate.Finally, 28 DEG C of each pipe, 150r/min shaking table culture 5 days. The MIC value of the metabolite of TRXY-46 is the minimum crude extract concentration for completely inhibiting notoginseng root rot bacterium visible growth.

As a result:

The percent inhibition that the metabolite of bacterial strain sophora tonkinensis Gapnep endogenetic fungus TRXY-46 grows notoginseng root rot bacterium mycelia It is as shown in table 3:

Table 3.

Note: positive control powder of carbendazim contains 50% carbendazim in table；* indicates that data pass through one-way analysis of variance in table LSD relatively after, the metabolite and positive control powder of carbendazim of bacterial strain TRXY-46 is under same concentrations, in P_0.05It is horizontal Upper tool significant difference.

The suppression result table that the metabolite of bacterial strain sophora tonkinensis Gapnep endogenetic fungus TRXY-46 grows notoginseng root rot bacterium mycelia Bright, the metabolite of bacterial strain sophora tonkinensis Gapnep endogenetic fungus TRXY-46 all has the mycelia growth of notoginseng root rot bacterium extraordinary Inhibitory effect, the metabolite of TRXY-46 presses down the percentage that notoginseng root rot bacterium F.solani mycelia grows as can be seen from Table 3 Rate processed is 90.10-99.83%, and compared with positive control powder of carbendazim, the metabolite of bacterial strain TRXY-46 is to Radix Notoginseng root-rot The inhibitory effect of germ F.solani mycelia growth is suitable with control.

Minimal inhibitory concentration such as 4 institute of table that sophora tonkinensis Gapnep endogenetic fungus TRXY-46 metabolite grows notoginseng root rot bacterium Show:

Table 4.

Processing	MIC(mg/ml)
			F.solani
Flusilazole	0.5
		TRXY-46	10

Note: positive control Flusilazole active constituent content is 400mg/mL in table.

The metabolite of bacterial strain sophora tonkinensis Gapnep endogenetic fungus TRXY-46 surveys the minimal inhibitory concentration that notoginseng root rot bacterium grows Test result shows that the metabolite of bacterial strain sophora tonkinensis Gapnep endogenetic fungus TRXY-46 all has stronger inhibition to notoginseng root rot bacterium It acts on, as can be seen from Table 4 minimum of the metabolite of sophora tonkinensis Gapnep endogenetic fungus TRXY-46 to notoginseng root rot bacterium F.solani Mlc is 10mg/ml, is 20 times of positive control.

As may be known from Table 3 and Table 4, very strong restraining epiphyte is contained in the metabolite of bacterial strain sophora tonkinensis Gapnep endogenetic fungus TRXY-46 Or antifungal ingredient, therefore, in the bionomic control of notoginseng root rot bacterium, bacterial strain sophora tonkinensis Gapnep endogenetic fungus TRXY-46 has Potentiality outstanding.

The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.

Claims (1)

1. a kind of application of metabolite of sophora tonkinensis Gapnep endogenetic fungus TRXY-46 in prevention and treatment notoginseng root rot, it is characterised in that: The classification naming of sophora tonkinensis Gapnep endogenetic fungus TRXY-46 is Rhexocercosporidium sp., deposit number: CGMCC No.10108；

The preparation method of the metabolite of the sophora tonkinensis Gapnep endogenetic fungus TRXY-46, operating procedure are as follows:

(1) sophora tonkinensis Gapnep endogenetic fungus TRXY-46 is inoculated in potato dextrose agar plane ware, being placed in temperature is It is cultivated 20 days at 28 DEG C, gained culture materials are cut into bulk and are transferred in sterile solid culture medium, are placed in 28 DEG C and ferment 60 days； Wherein, the sterile solid culture medium potato containing 400g, the dextrose and 20g sucrose of 20g；

(2) after fermentation, the methanol soaking fermentation object and ultrasound 40min with 2 times of fermentation material, is then filtered；

(3) step (2) are repeated twice, merges filtrate twice and medicinal extract is concentrated under reduced pressure into, obtains endogenetic fungus TRXY-46 fermentation The methanol crude extract of object, as sophora tonkinensis Gapnep endogenetic fungus TRXY-46 metabolite.