CN105483041A

CN105483041A - Application of sophora tonkinensis endogenetic bacterium B22 in prevention and treatment of panax notoginseng black spot

Info

Publication number: CN105483041A
Application number: CN201510979811.5A
Authority: CN
Inventors: 李良波; 姚裕群; 鲁璇; 黄荣韶
Original assignee: Guangxi University
Current assignee: Guangxi University
Priority date: 2015-12-23
Filing date: 2015-12-23
Publication date: 2016-04-13
Anticipated expiration: 2035-12-23
Also published as: CN105483041B

Abstract

The invention discloses sophora tonkinensis endogenetic bacterium B22. The sophora tonkinensis endogenetic bacterium B22 has the type name of Microbacterium sp. B22, a 16S rDNA gene sequence table of the Microbacterium sp. B22 is as shown in SEQ ID NO. 1, and the Microbacterium sp. B22 is collected by the General Microorganisms Center of China Committee for Culture Collection of Microorganisms, which is located at Institute of Microbiology, Chinese Academy of Sciences, 3#, 1# Courtyard, Beichen West Road, Chaoyang District, Beijing, on September 29, 2015 and has the collection number of CGMCC No. 11463. According to the sophora tonkinensis endogenetic bacterium B22, the endogenetic bacterium Microbacterium sp. B22 is obtained through being separated from roots of a medicinal plant, i.e., Sophora tonkinensis and carrying out screening for the first time and has a powerful inhibiting action on Alternaria panax Whetzel, and thus a broad application prospect is brought to the field of biological control on panax notoginseng fungal diseases.

Description

The application of sophora tonkinensis Gapnep endogenetic bacteria B22 in control Alternaria panax

Technical field

The present invention relates to biological technical field, the particularly application of a kind of sophora tonkinensis Gapnep endogenetic bacteria B22 in control Alternaria panax.

Background technology

The control of modern agriculture to Plant diseases is overly dependent upon the use of chemical pesticide, discharging of a large amount of chemical pesticide not only has long-term destruction to ecotope, also cause quality of agricultural product to decline, pesticide residue exceed standard, the resistance of pathogenic bacteria and to problems such as people and animals are harmful.Find safer, effective pest control method to be significant, utilizing biological method to carry out controlling plant diseases can effectively solve the problem.

Pseudo-ginseng (PanaxnotoginsengF.H.chen) has another name called pseudo-ginseng, invaluable etc., and having promoting blood circulation and removing blood stasis, subduing swelling and relieving pain effect significantly, is a kind of Chinese tradition rare medicinal herbs.Be that the Chinese patent medicine that main raw material is made is as wide-spread in " Yunnan white powder " and " Pien Tze Huang " etc. with pseudo-ginseng.In recent years, growing to the raw-material demand of pseudo-ginseng.But the fungal disease in cultivation has had a strong impact on Panax notoginseng Growth.At present, on notoginseng planting, control fungal disease depends on chemical pesticide unduly, and the use of a large amount of chemical pesticide not only have impact on the quality of pseudo-ginseng, also result in a large amount of pesticide residue of pseudo-ginseng.

Alternaria panax can infect pseudo-ginseng plant and underground root system, be injured heavier with stem, leaf, floral axis, in brown web rot shape after root infection, the four seasons all can occur, pseudo-ginseng garden temperature of shed is high, high humidity, improper fertilization all likely makes disease spread to increase the weight of, general long-term sickness rate between 5%-20%, up to more than 60% time serious.Its symptom is mostly caused by Alternaria panax bacterium AlternariapanaxWhetzel (being called for short A.panax) harm.

This fungal disease is the one of the main reasons causing pseudo-ginseng quality product and production declining for many years.In traditional Genuine producing area of Guangxi pseudo-ginseng, due to the meteorological conditions of low altitude area and high temperature and humidity, this disease is simultaneous often, and a large amount of underproduction causing pseudo-ginseng are even had no harvest, and brings huge financial loss to plantation family.In order to control this fungal disease, a large amount of chemical pesticides is used, and cause a large amount of pesticide residue of pseudo-ginseng product, serious pollutes environment, has threatened the health of people greatly.Therefore, it is very urgent and necessary for carrying out the Sustainable development of biological control pseudo-ginseng fungal disease to pseudo-ginseng industry.Filter out for this reason and can the Antagonistic Fungi of simultaneously this disease of antagonism be significant, screening and then to develop Antagonistic Fungi be also effectively control one of the most potential prophylactico-therapeutic measures of pseudo-ginseng fungal disease.

The information being disclosed in this background technology part is only intended to increase the understanding to general background of the present invention, and should not be regarded as admitting or imply in any form that this information structure has been prior art that persons skilled in the art are known.

Summary of the invention

The object of the present invention is to provide the application of a kind of sophora tonkinensis Gapnep endogenetic bacteria B22 in control Alternaria panax, thus the Alternaria panax occurred in energy effectively preventing notoginseng planting process, thus improve output and the quality of pseudo-ginseng.

For achieving the above object, technical scheme provided by the invention is as follows:

A kind of sophora tonkinensis Gapnep endogenetic bacteria B22, the Classification And Nomenclature of sophora tonkinensis Gapnep endophyte B22 is microbacterium (Microbacteriumsp.) B22, the 16SrDNA gene order table of bacterial strain is as described in SEQIDNO.1, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica, preservation date: on 09 29th, 2015, preserving number: CGMCCNo.11463.

Preferably, the preparation method of described sophora tonkinensis Gapnep endogenetic bacteria B22 meta-bolites is: be inoculated in NB liquid nutrient medium by sophora tonkinensis Gapnep endogenetic bacteria B22, be placed in that temperature is 28 DEG C, rotating speed is that 130r/min condition bottom fermentation cultivates 10 days, gained fermented product is after concentrating under reduced pressure drying, add the methyl alcohol of fermented product 2 times and ultrasonic 40min, then centrifugal, get the methanol crude extract that supernatant liquor concentrating under reduced pressure obtains strain fermentation thing, be sophora tonkinensis Gapnep endogenetic bacteria B22 meta-bolites.

Preferably, described sophora tonkinensis Gapnep endogenetic bacteria B22 is inoculated in containing 1000mlNB liquid nutrient medium.

Preferably, described NB liquid nutrient medium is containing beef extract 3g, yeast extract paste 1g, peptone 5g, sucrose 10g, and Medium's PH Value is 7.0.

The application of meta-bolites in control Alternaria panax of gained sophora tonkinensis Gapnep endogenetic bacteria B22 is prepared as above-mentioned.

Compared with prior art, the present invention has following beneficial effect:

The present invention first from the root of medicinal plant sophora tonkinensis Gapnep separation screening to strain sophora tonkinensis Gapnep endogenetic bacteria (Burkholderiasp.) B22, this bacterium has very strong restraining effect to Alternaria panax bacterium, and the field of biological control for pseudo-ginseng fungal disease brings wide application prospect.

Accompanying drawing explanation

Fig. 1 is the dull and stereotyped opposite culture of sophora tonkinensis Gapnep endogenetic bacteria B22 of the present invention and Alternaria panax, and wherein F is Alternaria panax bacterium (AlternariapanaxWhetzel), a is blank, and b is experimental group.

Fig. 2 is sophora tonkinensis Gapnep endogenetic bacteria B22 strain morphology feature of the present invention, and wherein, a1 is colonial morphology, and b1 is thalli morphology.

Fig. 3 is the phylogenetic tree that sophora tonkinensis Gapnep endogenetic bacteria B22 bacterial strain builds based on 16SrDNA gene order.

Fig. 4 is to the inhibition of the mycelial growth of Alternaria panax bacterium according to the meta-bolites of bacterial strain sophora tonkinensis Gapnep endogenetic bacteria B22 of the present invention; Wherein the 1st, the 5th is that namely the PDA of pastille is not dull and stereotyped for blank; 2nd, the 3rd, the 4th is positive control powder of carbendazim; 6th, the 7th, the 8th is the meta-bolites of B22 bacterial strain, and concentration is respectively 2mg/ml, 4mg/ml, 8mg/ml; L is Alternaria panax bacterium AlternariapanaxWhetz.

Embodiment

Be described in detail below in conjunction with embodiment, but be to be understood that protection scope of the present invention not by the restriction of embodiment.The experimental technique used in following embodiment if no special instructions, is ordinary method.The material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.Methyl alcohol is commercial analytical pure methyl alcohol.2XTagMasterMix is purchased from precious biotechnology company limited (Takara), and Primer-1, Primer-2 are by Hua Da gene chemical synthesis.

Embodiment 1

The separation of bacterial strain, screening and qualification

One, the separation of bacterial strain

For examination material: the wild sophora tonkinensis Gapnep picking up from In Limestone Area, Tiandeng County, Guangxi.

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

Substratum: 1000mlNA substratum: beef extract 3g, yeast extract paste 1g, peptone 5g, sucrose 10g, agar 15g, pH7.0.

Surface sterilization: by long for 6-8cm, the wide sophora tonkinensis Gapnep root running water 30min for 1-2cm fresh and healthy are to rinse silt, then the sophora tonkinensis Gapnep root rinsed with sterile water 2 times will cleaned, air-dry surface-moisture, moving on to Bechtop, aseptically, is 75% alcohol immersion 1min by sophora tonkinensis Gapnep root volumetric concentration, rinsed with sterile water 2 times, clorox (available chlorine 1%) soaks 2min, aseptic washing 3 times, and it is for subsequent use that aseptic thieving paper blots surface.

The separation of bacterial strain, purifying: the sophora tonkinensis Gapnep root that surface sterilization is good, under aseptic condition, epidermis is scraped off with aseptic wood chip, the two ends of sophora tonkinensis Gapnep root are cut off with aseptic secateurs, remaining part aseptic pocket knife and aseptic nipper separate xylem and phloem, get the tissue block that 0.5cm is long respectively, shred and add sterilized water grinding with aseptic secateurs, add sterilized water after grinding be fully settled to 5ml and leave standstill 10min, get 0.5ml supernatant liquor gradient dilution 10 ~ 10 ⁵doubly, get 100 μ L diluents respectively and be applied on NA flat board, each weaker concn repeats 3 times, gets last rinsing liquid and is coated on NA flat board, as negative control.NA flat board is placed in incubator 28 DEG C of constant temperature culture 24 ~ 96h, and picking different shape bacterium colony is rule purifying repeatedly, and by the bacterial strain of purifying, namely sophora tonkinensis Gapnep endogenetic bacteria 20% glycerine is preserved.

The sophora tonkinensis Gapnep endogenetic bacteria be separated to is inoculated on LA flat board, stand-by after cultivating 24h at 28 DEG C.Alternaria panax bacterium is inoculated in PDA flat board, cultivates after 3 days stand-by at 28 DEG C.

Two, screen

Adopt dull and stereotyped face-off method to the primary dcreening operation carrying out thalline bacteriostatic activity for examination sophora tonkinensis Gapnep endogenetic bacteria.

First, under aseptic condition, with sterilizing punch tool, Alternaria panax bacterium is made 6mm bacterium cake; In processes, Alternaria panax bacterium bacterium cake is transferred to the culture dish central authorities containing NA, then in the position of distance bacterium cake 2cm, the sophora tonkinensis Gapnep endogenetic bacteria bacterial strain be separated to is connected a bacterium line, and every strain endogenetic bacteria repeats 3 wares; In control group, only connect Alternaria panax bacterium bacterium cake, do not connect sophora tonkinensis Gapnep endogenetic bacteria, repeat 3 wares.Then, cultivate at 28 DEG C, periodic observation.When fungi covers with culture dish in control group, in treatment group, measure the growth radius of Alternaria panax bacterium between Alternaria panax Jun Junbing center to sophora tonkinensis Gapnep endogenetic bacteria line center for process growth radius; In control group, the growth radius of Alternaria panax bacterium is contrast growth radius.Finally, according to following formula, calculate bacteriostasis rate:

Contrast increment=contrast growth radius-bacterium cake radius

Process increment=process growth radius-bacterium cake radius

Result obtains the 3 strains antagonism sophora tonkinensis Gapnep endogenetic bacteria bacterial strain all very strong to Alternaria panax bacterium restraining effect altogether, and wherein a strain name is called B22, is 57% to the inhibiting rate of Alternaria panax bacterium.

Three, identify

(1) strain morphology feature

Colony morphology characteristic is observed: by inoculation to be identified on NA substratum, is placed in 28 DEG C and cultivates 24h, observe the colony morphology characteristic of bacterial strain.

Morphological features is observed: get the above-mentioned bacterial strain cultivating 24h on NA substratum and carry out gramstaining and microscopy, by the photomicrography of viewed form result, as shown in Figure 2.

(2) bacterial strain 16SrDNA sequence and phylogenetic analysis thereof

The preparation of DNA profiling:

Reagent: (1) lysis buffer: Tris-Ac (pH7.8) 40mM, NaAc20mM, EDTA1mM, SDS1% (w/v);

(2) 5MNaCl solution.

DNA extraction:

A 1.5ml overnight bacterial nutrient solution culture is placed in Eppendorf tube (EP pipe) by (), the centrifugal 0.5min of 12000rpm, abandoning supernatant, retains throw out;

B () adds 400 μ l lysis buffers in throw out, repeatedly blow and beat make it resuspended with suction pipe;

C () adds 200ul5MNaCl, fully mix, the centrifugal 10min of 12000rpm, gets 600 μ l supernatant liquors;

D () adds isopyknic phenol/chloroform (1:1), mixing, and centrifugal (12000rpm, 10min), proceeds in another clean EP pipe by supernatant liquor;

Add equal-volume chloroform in e supernatant liquor that () obtains in step (d), mixing, centrifugal (12000rpm, 10min), proceeds in another clean EP pipe by supernatant liquor;

F () adds isopyknic Virahol in gained supernatant liquor in step (e), mixing, is placed in room temperature 10min, centrifugal (12000rpm, 15min), abandoning supernatant, retains throw out;

G () is 70% washing with alcohol step (f) gained throw out by volumetric concentration, dry, be DNA;

H DNA dry in step (g) is dissolved in 30 μ l distilled water (ddH by () ₂o) in ,-20 DEG C of preservations.

Pcr amplification 16SrDNA sequence:

(1) PCR instrument: ABI3730-XLDNA sequenator (AppliedBiosystems, USA);

(2) amplimer: 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') is as shown in SEQIDNO.2, and 1492R (5 '-GGTTACCTTGTTACGACT-3 ') is as shown in SEQIDNO.3;

(3) amplification system:

16SrDNA sequence PCR amplification system is as shown in table 1:

Table 1.

Reactant	Application of sample amount
		2X TagMasterMix	25μL
Primer-1	1μL
		Primer-2	1μL
Template DNA	0.5μL
		ddH ₂O	22.5μL
Reaction cumulative volume	50μL

Note: the TemplateDNA in table 1 is that above-mentioned steps (h) gained DNA makes DNA profiling.

PCR reaction conditions is as shown in table 2:

Table 2.

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

The electrophoresis detection of pcr amplification product:

Deposition condition is the sepharose (containing Goldview5 μ l/100ml) of 1%, 1 × TBE electrophoretic buffer, 90V electrophoresis 1 hour, and PCR primer applied sample amount is 3 μ L, point sample after mixing with 1 μ LLoadingdye.Observations under 254nm ultraviolet, with the DL1000DNAMarker of TaKaRa company for nucleic acid standard molecular weight object of reference, determines expanding fragment length.Amplified production band should on the position of standard substance 400-700bp.

PCR primer purifying and order-checking: undertaken by Shenzhen Huada Genetic Technology Co., Ltd.

The structure of systematic evolution tree:

The 16SrDNA sequence of the bacterium measured in surveyed 16SrDNA sequence and GenBank gene pool is compared, downloads correlated series according to comparison result.Systems analysis is carried out and constructing system evolutionary tree, as shown in Figure 3 by the adjacent algorithm (Neighbor-joiningNJ) of MEGA6.0.

Result:

(1) strain morphology feature

Colony morphology characteristic: bacterial strain bacterium colony is in faint yellow on NA flat board, and smooth surface, protuberance, neat in edge, circular, on NA flat board, cultivation 5 days diameters are for 3-4mm is as shown in a1 in Fig. 2.

Morphological features: Gram-negative is negative staining, without gemma, thalline direct rod shape, size is (0.32 ~ 0.64) μm × (0.84 ~ 1.30) μm, as shown in b1 in Fig. 2.

(2) bacterial strain 16SrDNA sequence and phylogenetic analysis thereof

Utilize primer 2 7F and 1492R, from strain gene group DNA, amplify the fragment of a 1300-1400bp size, through order-checking and by sequencing result by BLASTn comparison in GenBank.Result shows, bacterium in bacterial strain sophora tonkinensis Gapnep endophyte B22 and Microbacteriu has very high base sequence similarity, therefore the reference strain sequence of Microbacteriu in all GenBank is downloaded, for Phylogenetic Analysis, using the Agromycesindicus (NR108908) in Agromyces and Agromycesmediolanus (NR117879) as outer group.In the systematic evolution tree built, sophora tonkinensis Gapnep endophyte B22 and Microbacteriumsp.FSBSY9 (KJ184984) gets together and forms holding strength is the end branch of 100%.Base similarity system design result shows, sophora tonkinensis Gapnep endophyte B22 and Microbacteriumsp.FSBSY9 (KJ184984) has the difference of 15 bases, and sequence similarity is 98.9%.Comprehensive morphological and molecular biological characteristics, be initially identified as Microbacteriumsp. by bacterial strain.

Embodiment 2

Metabolite is to the restraining effect of Alternaria panax bacterium

One, the fermentation culture of bacterial strain B22 and the extraction of bacterial strain B22 meta-bolites

Sophora tonkinensis Gapnep endophyte B22 is inoculated in containing 1000mlNB liquid nutrient medium (beef extract 3g, yeast extract paste 1g, peptone 5g, sucrose 10g, pH7.0) in 2 liters of Erlenmeyer flasks, be placed in the shaker fermentation that temperature is 28 DEG C, rotating speed is 130r/min and cultivate 10 days, gained fermented product is after concentrating under reduced pressure drying, add the methyl alcohol of fermented product 2 times and use ultrasonic echography 40min, ultrasonic carry out centrifugal, get the methanol crude extract that centrifugal rear gained supernatant liquor concentrating under reduced pressure obtains strain fermentation thing, be sophora tonkinensis Gapnep endogenetic bacteria B22 meta-bolites.

Two, sophora tonkinensis Gapnep endogenetic bacteria B22 meta-bolites is to the restraining effect of the mycelial growth of Alternaria panax bacterium

The B22 meta-bolites of bacterial strain is measured to the inhibit activities of Alternaria panax bacterium mycelial growth by mycelial growth method.The pastille B22 meta-bolites of bacterial strain and powder of carbendazim (positive control) made respectively containing 2mg/mL, 4mg/mL, 8mg/mL concentration B22 meta-bolites is dull and stereotyped.Aseptically, with punch tool, pathogenic fungi is made 6mm bacterium cake, it is process that Alternaria panax bacterium bacterium cake is received the dull and stereotyped central authorities of each pastille, and the Alternaria panax bacterium bacterium cake connect with not dull and stereotyped central authorities of pastille is for negative control, in triplicate, all flat boards are placed in 28 DEG C of cultivations for all process and contrast.The Field information concentration that drug level presses powder of carbendazim is arranged.When negative control covers with culture dish, measure the growth diameter of contrast bacterium colony and process bacterium colony respectively, and according to following formula, calculate inhibiting rate:

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

Process increment=process growth diameter-bacterium cake diameter

Three, sophora tonkinensis Gapnep endogenetic bacteria B22 meta-bolites is to the minimal inhibitory concentration of Alternaria panax bacterium

With the B22 meta-bolites of broth dilution method determination bacterial strain to the minimal inhibitory concentration of Alternaria panax bacterium.PDA is cultivated the Alternaria panax bacterium pure culture biscuits involvng inoculation of 5 days in the PDB liquid nutrient medium containing 0.2% tween 80 (v/v), 28 DEG C, 150r/min shaking table cultivates 7 days, then culture is forwarded to triangular flask, and the aseptic double-distilled water poured into containing 0.2% tween 80, stir 30min with magnetism stick, solution sterile gauze is filtered, obtain spores solution, and with blood counting chamber, spore concentration is adjusted to every milliliter 10 ⁴individual spore is for subsequent use.The meta-bolites 1%DMSO of bacterial strain B22 is dissolved into the meta-bolites concentration for the treatment of of 80mg/mL, getting 5 sterile test tube is sequentially arranged on test-tube stand, be numbered 1,2,3,4,5 respectively, often pipe adds the aseptic double-distilled water of 1ml containing 0.2% tween 80 respectively, is then added by the bacterial strain methanol crude extract solution of 1ml80mg/mL in the test tube being numbered 1 and also in each pipe, carries out twice serial dilution successively.The above-mentioned gained spores solution (10 of 1ml ⁴individual/ml) add in each pipe respectively, thus obtain final B22 meta-bolites concentration for the treatment of: 20mg/ml (numbering 1 test tube), 10mg/ml (numbering 2 test tube), 5mg/ml (numbering 3 test tube), 2.5mg/ml (numbering 4 test tube), 1.25mg/ml (numbering 5 test tube).The 8mg/mL Flusilazole of 1%DMSO and 1ml of 1ml replaces B22 meta-bolites to perform above-mentioned treating processes respectively and as negative control and positive control, all process and contrast are in triplicate.Finally, each pipe 28 DEG C, 150r/min shaking table cultivates 5 days.The MIC value of B22 meta-bolites is suppress the minimum crude extract concentration of Alternaria panax bacterium visible growth completely.

Result:

The percent inhibition of bacterial strain sophora tonkinensis Gapnep endophyte B22 meta-bolites to Alternaria panax bacterium mycelial growth is as shown in table 3:

Table 3.

Note: the positives contrast powder of carbendazim of table 3 is containing 50% derosal; After in table, * represents that LSD that data pass through one-way analysis of variance is relatively, bacterial strain B22 meta-bolites and positive control powder of carbendazim under same concentrations, at P _0.05tool significant difference in level.

The suppression result of meta-bolites to Alternaria panax bacterium mycelial growth of bacterial strain sophora tonkinensis Gapnep endophyte B22 shows, the meta-bolites of bacterial strain sophora tonkinensis Gapnep endophyte B22 all has extraordinary inhibition to the mycelial growth of Alternaria panax bacterium, and the percent inhibition of meta-bolites to Alternaria panax bacterium A.panax mycelial growth of sophora tonkinensis Gapnep endophyte B22 is 82.8-100% as can be seen from Table 3.Compare with positive control powder of carbendazim, the meta-bolites of bacterial strain sophora tonkinensis Gapnep endophyte B22 is better than contrast to the inhibition of Alternaria panax bacterium A.panax mycelial growth.

The minimal inhibitory concentration of meta-bolites to Alternaria panax bacteria growing of bacterial strain B22 is as shown in table 4:

Table 4.

Process	MIC(mg/ml)
			A.panax
Fluzilazol	0.44
		B22	1.25

Note: showing positives contrast fluzilazol is pure compound, and purity is 99.99%.

The minimal inhibitory concentration test result of meta-bolites to Alternaria panax bacteria growing of bacterial strain sophora tonkinensis Gapnep endophyte B22 shows, the meta-bolites of bacterial strain sophora tonkinensis Gapnep endophyte B22 all has stronger restraining effect to Alternaria panax bacterium, the minimal inhibitory concentration of meta-bolites to Alternaria panax bacterium A.panax of sophora tonkinensis Gapnep endophyte B22 is 1.25mg/ml as can be seen from Table 4, is 2.8 times of positive control.

As may be known from Table 3 and Table 4, contain the composition of very strong antifungal or fungicidal in the meta-bolites of bacterial strain sophora tonkinensis Gapnep endophyte B22, therefore, in the bionomic control of Alternaria panax bacterium, bacterial strain sophora tonkinensis Gapnep endophyte B22 has outstanding potentiality.

The aforementioned description to concrete exemplary of the present invention is to illustrate and the object of illustration.These descriptions not want the present invention to be defined as disclosed precise forms, and obviously, according to above-mentioned instruction, can much change and change.The object selected exemplary embodiment and describe is to explain certain principles of the present invention and practical application thereof, thus those skilled in the art can be realized and utilize various different exemplary of the present invention and various different selection and change.Scope of the present invention is intended to limited by claims and equivalents thereof.

Claims

1. a sophora tonkinensis Gapnep endogenetic bacteria B22, is characterized in that: the Classification And Nomenclature of sophora tonkinensis Gapnep endophyte B22 is microbacterium (Microbacteriumsp.) B22, preserving number: CGMCCNo.11463.

2. the preparation method of the meta-bolites of sophora tonkinensis Gapnep endogenetic bacteria B22 as claimed in claim 1, it is characterized in that: sophora tonkinensis Gapnep endogenetic bacteria B22 is inoculated in NB liquid nutrient medium, be placed in that temperature is 28 DEG C, rotating speed is that 130r/min condition bottom fermentation cultivates 10 days, gained fermented product is after concentrating under reduced pressure drying, add the methyl alcohol of fermented product 2 times and ultrasonic 40min, then centrifugal, get the methanol crude extract that supernatant liquor concentrating under reduced pressure obtains strain fermentation thing, be sophora tonkinensis Gapnep endogenetic bacteria B22 meta-bolites.

3. the preparation method of meta-bolites according to claim 2, is characterized in that: described sophora tonkinensis Gapnep endogenetic bacteria B22 is inoculated in containing 1000mlNB liquid nutrient medium.

4. the preparation method of meta-bolites according to claim 2, is characterized in that: described NB liquid nutrient medium is containing beef extract 3g, yeast extract paste 1g, peptone 5g, sucrose 10g, and Medium's PH Value is 7.0.

5. the application of meta-bolites in control Alternaria panax preparing gained sophora tonkinensis Gapnep endogenetic bacteria B22 as claim 2.