CN110438011A - Superfine branch spore is mould and its promotes the purposes of Radix Salviae Miltiorrhizae root system effective component synthesis - Google Patents
Superfine branch spore is mould and its promotes the purposes of Radix Salviae Miltiorrhizae root system effective component synthesis Download PDFInfo
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Abstract
The invention discloses a kind of Radix Salviae Miltiorrhizae endogenetic fungus DF11, are the mould Cladosporium tenuissimum of superfine branch spore, and deposit number is CGMCC No.16883.The present invention further simultaneously discloses the purposes of above-mentioned Radix Salviae Miltiorrhizae endogenetic fungus DF11: promoting the synthesis of Radix Salviae Miltiorrhizae root system effective component, promotes the growth of Radix Salviae Miltiorrhizae root system；The effective component is tanshinone substance, danshinolic acid substance.Radix Salviae Miltiorrhizae endogenetic fungus of the invention can effectively promote Radix Salviae Miltiorrhizae root system development by co-culturing with Radix Salviae Miltiorrhizae plant, can also effectively facilitate the biosynthesis of effective component in red sage, has biggish application value.
Description
Technical field
The present invention relates to a kind of Radix Salviae Miltiorrhizae root system endogenetic fungus and application thereof --- for promoting Radix Salviae Miltiorrhizae root system effective component to close
At.
Background technique
Salviamiltiorrhizabung (scientific name: Salvia miltiorrhiza Bge.) is the root of Lamiaceae plant Radix Salviae Miltiorrhizae.With promoting blood circulation
Stagnation resolvation, the multiple efficacies such as cool blood to disappear carbuncle, analgesic, relieving restlessness and restlessness.According to its traditional effect, Radix Salviae Miltiorrhizae is widely used in heart and brain blood in the modern times
The treatment of pipe disease, and obtain significant curative effect.The medicinal ingredient of Radix Salviae Miltiorrhizae includes water-soluble danshinolic acid class and fat-soluble Radix Salviae Miltiorrhizae
Ketones component.Water-soluble phenolic acid compound mainly has salviandic acid A, B, C, caffeic acid, danshensu etc., has antithrombotic shape
At, it is anti-oxidant, protection cell, anticoagulation and adjust blood lipid the effects of.Tanshinone component mainly includes Tanshinone I, dihydro pellet
Join ketone I, Tanshinone I I A, Tanshinone I I B, Cryptotanshinone, hydroxyl tanshinone etc., there is anti-inflammatory, antibacterial and improves blood circulation
And other effects.
Since the market demand of Radix Salviae Miltiorrhizae is big, and wild resource is increasingly reduced, and cultivar degenerates serious.Artificial introducing and planting
In the presence of the influence for introducing a fine variety the factors such as difficult and environmental constraints, the content of secondary metabolite is unstable in plant, the quality of medicinal material
It cannot be guaranteed.Therefore, how using new technology and means activated product content is greatly improved, it is more effective, sustainable and low
Active chemical in the acquisition Radix Salviae Miltiorrhizae of cost, it has also become the important content and development side of Radix Salviae Miltiorrhizae resource and activated product research
To.
Invention " the Radix Salviae Miltiorrhizae endophyte and its use with induction tanshin polyphenolic acid B summation of the patent No. 201510178706.1
Pseudomonas psychrotolerans LG4, deposit number are disclosed on the way " are as follows: CCTCC NO:M 2015085 can promote
The accumulation of phenolic acid (tanshin polyphenolic acid B) content in Hairy Root Cultures of Salvia miltiorrhiza.The invention of the patent No. 201510176190.7 " has induction
The endophyte and application thereof of Hairy Root Cultures of Salvia miltiorrhiza phenolic acid summation is Olivibacter soli LG3, deposit number are as follows:
CCTCC NO:M2015084.Invention " the Radix Salviae Miltiorrhizae endophyte with induction phenolic acid summation of the patent No. 201510175259.4
And application thereof be Pseudomonas chlororaphis LG1 (Pseudomonas chlororaphis LG1), deposit number are as follows: CCTCC NO:M
2015082。
2013100508029 invention " the Cladosporium fungi of one plant of raising Radix Salviae Miltiorrhizae yield and total phenol acid content " informs bacterium
Strain 11673-58, deposit number are the branch spore (Cladosporium sp.) of CGMCC No.6608, through planting with Radix Salviae Miltiorrhizae seedling symbiosis
Train the 4-6 month, the content of the yield of Radix Salviae Miltiorrhizae and salvianolic acid and tanshin polyphenolic acid B when can improve harvest time.
" influence of the endogenetic fungus Cladosporium Cladosporium sp. to Salvia miltiorrhiza Growth and danshinolic acid content ", informs
SM58 bacterial strain (CGMCC No.6608) not only has significant growth promoting function to the plant height, root long and biomass of Radix Salviae Miltiorrhizae tissue-cultured seedling
(P < 0.01), and also have remarkable result to the raising of danshinolic acid active constituent content and root dry weight that Radix Salviae Miltiorrhizae is planted in potting and field
(P<0.01).It is compared with a control, connects bacterium group field and plant the root dry weight of Radix Salviae Miltiorrhizae, total phenol acid content, the content of salviandic acid A and be respectively increased
68%, 47%, 11%.
The purposes for the Radix Salviae Miltiorrhizae endophyte that above-mentioned patent or paper are informed is to be related to improving Tanshin Water-soluble Ingredient ---
Phenolic acid content is not directed to the promotion of liposoluble constituent tanshinone content.
Application number 201611096389X " a kind of endogenetic fungus for improving Radix Salviae Miltiorrhizae yield and its active constituent content and its
Using " a kind of chaetomium globosum D68 (Chaetomium globosum) is provided, deposit number is CGMCC No.12622；Also into
One step discloses Radix Salviae Miltiorrhizae endogenetic fungus in the purposes for improving host's Radix Salviae Miltiorrhizae biomass and tanshinone and liposoluble ingredient content.It should
Radix Salviae Miltiorrhizae endogenetic fungus is used from Lamiaceae Salvia platymiscium Radix Salviae Miltiorrhizae Salvia miltiorrhiza Bunge plant living body
The separation of endogenetic fungus separating and purifying technology obtains.
As the common sense of the industry, chaetomium globosum (Chaetomium globosum) and the mould (Cladosporium of branch spore
Sp) belong to different kinds in classification, and the normal usage of chaetomium globosum (C.globosum) is applied to biological control
Beneficial to fungi；And many species of Cladosporium (Cladosporium) are common phytopathogens, also have many species to be
Plant endogenesis or fungal component will not cause obvious disease；Therefore chaetomium globosum (C.globosum) purposes with
The purposes of Cladosporium sp. does not have comparativity.
Based on finding research, endophyte of plant has most important on to host's root system development and secondary metabolite accumulation
Effect.Currently, the research of medicinal plant endophyte is still in the primary stage, so far about plant source bioactive process can be generated
It is less to close the report that the endogenetic fungus elicitor of object influences host plant secondary metabolism, separation endophyte is simultaneously from medicinal plant
And fermenting and producing is carried out using endophyte of plant, or be inoculated with specific microorganism in medicinal plant to obtain certain important days
Right drug or certain genunie medicinal materials, will be deficient, raw to medicine source caused by certain medicinal plant slow growths, resource scarcity etc. is solved
State, which destroys problem, directive significance.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of Radix Salviae Miltiorrhizae endogenetic fungus and application thereof, can promote in Radix Salviae Miltiorrhizae root system
The synthesis of tanshinone.
It is that superfine branch spore is mould in order to solve the above technical problem, the present invention provides a kind of Radix Salviae Miltiorrhizae endogenetic fungus DF11
Cladosporium tenuissimum, deposit number are CGMCC No.16883.
The present invention goes back while providing the purposes of above-mentioned Radix Salviae Miltiorrhizae endogenetic fungus DF11: promoting the conjunction of Radix Salviae Miltiorrhizae root system effective component
At, promote Radix Salviae Miltiorrhizae root system growth.
The improvement of purposes as Radix Salviae Miltiorrhizae endogenetic fungus DF11 of the invention: the effective component be tanshinone substance,
Danshinolic acid substance；
The tanshinone substance includes: Cryptotanshinone, dihydrotanshinone, Tanshinone I, tanshinone IIA,
The danshinolic acid substance includes Rosmarinic acid, tanshin polyphenolic acid B.
The present invention goes back while providing a kind of method for improving active constituent content in Radix Salviae Miltiorrhizae: by Radix Salviae Miltiorrhizae endogenetic fungus DF11
It is seeded to Radix Salviae Miltiorrhizae aseptic seedling root, co-cultures Radix Salviae Miltiorrhizae aseptic seedling and Radix Salviae Miltiorrhizae endogenetic fungus DF11 in (25 ± 0.5) DEG C；When culture
Between be (60 ± 5) day.
Improvement as the method for active constituent content in raising Radix Salviae Miltiorrhizae of the invention: it is connect in each Radix Salviae Miltiorrhizae aseptic seedling root
Touch the bacteria suspension 1mL that Radix Salviae Miltiorrhizae endogenetic fungus DF11 is inoculated at matrix；
The concentration of bacteria suspension is 106CFU/mL。
The Radix Salviae Miltiorrhizae endogenetic fungus is from Lamiaceae Salvia platymiscium Radix Salviae Miltiorrhizae (Salvia miltiorrhiza Bunge) root
It is obtained in system using the separation of endogenetic fungus separating and purifying technology, it is mould to be accredited as superfine branch spore through microbial taxonomy
(Cladosporium tenuissimum)。
Culture medium used in the present invention includes:
A martin substratum formula: peptone 5.0g) is improved；Dipotassium hydrogen phosphate 1.0g；Yeast extract powder 2.0g；Sulfuric acid
Magnesium 0.5g；Glucose 20.0g；Distilled water 1000ml；PH is natural；It is mainly used for the separation and purifying of endogenetic fungus.
B) PDA solid potato culture medium formula: potato 200g, glucose 20g, deionized water 1000mL, pH 7.2；
It is mainly used for the activation and culture of endogenetic fungus.
C) Radix Salviae Miltiorrhizae Aseptic seedling culture base: MS culture medium, pH 5.8；The expansion for being mainly used for Radix Salviae Miltiorrhizae aseptic seedling is numerous.
D) endogenetic fungus and Radix Salviae Miltiorrhizae co-culture system: potting soil matrix is fertile soil: perlite: vermiculite=1:1:l is mixed
Close cultivation matrix.Holder: 1300ml PP plastics tissue culture bottle.Hot-house culture temperature: 25 DEG C.It is mainly used for endogenetic fungus to return
Connect the interaction experiment of aseptic seedling.
Tanshinone and phenolic acid effective constituents of the invention includes: Cryptotanshinone, dihydrotanshinone, Tanshinone I, Radix Salviae Miltiorrhizae
Ketone IIA, Rosmarinic acid, tanshin polyphenolic acid B.
Cryptotanshinone, also known as: cryptotanshinone, English name: Cryptotanshinone, molecular formula: C19H20O3, CAS:
35825-57-1, chemical structural formula are as follows:
Dihydrotanshinone, English name: Dihydrotanshinone, molecular formula: C18H14O3, CAS:20958-18-3, chemistry
Structural formula is as follows:
Wherein, Tanshinone I, English name: Tanshinone I, molecular formula: C18H12O3, CAS:568-73-0, chemical structure
Formula is as follows:
Tanshinone IIA, English name: Tanshinone IIA, molecular formula: C19H18O3, CAS:568-72-9, chemical structural formula
It is as follows:
Rosmarinic acid, English name: Rosmarinic acid, molecular formula: C18H16O8, CAS:20283-92-5, chemical structure
Formula is as follows:
Tanshin polyphenolic acid B, English name: Salvianolic acid B, molecular formula: C36H30O16, CAS:115939-25-8, chemistry
Structural formula is as follows:
The present invention has following technical advantage:
Radix Salviae Miltiorrhizae endogenetic fungus of the present invention can effectively promote Radix Salviae Miltiorrhizae root system hair by co-culturing with Radix Salviae Miltiorrhizae plant
It educates, can also effectively facilitate the biosynthesis of effective component in red sage, there is biggish application value.
Detailed description of the invention
Specific embodiments of the present invention will be described in further detail with reference to the accompanying drawing.
The aspect graph of Fig. 1 Radix Salviae Miltiorrhizae endogenetic fungus of the present invention；
A, B is aspect graph of the Radix Salviae Miltiorrhizae endogenetic fungus of the present invention on PDA plate culture medium, and wherein A is DF11 bacterium colony
Positive form, B are the form at the bacterium colony back side；
C is Radix Salviae Miltiorrhizae endogenetic fungus of the present invention in 10 × 100 times of lower aspect graphs of optical microscopy.
Radix Salviae Miltiorrhizae plant root development situation after Fig. 2 Radix Salviae Miltiorrhizae endogenetic fungus DF11 processing 60 days of the present invention, A is control
(CK), B is endogenetic fungus DF11 processing group of the present invention.
Fig. 3 is the comparison of CK (control group) and DF11 (endogenetic fungus processing group of the present invention).
Fig. 4 be CK (control group) and DF11 (endogenetic fungus processing group of the present invention) Cryptotanshinone after 60 days, dihydrotanshinone,
The comparison of the content of Tanshinone I, tanshinone IIA, Rosmarinic acid, tanshin polyphenolic acid B.
Fig. 5 is ITS sequence segment.
Specific embodiment
The present invention is described further combined with specific embodiments below, but protection scope of the present invention is not limited in
This:
The isolated culture method of embodiment 1, Cladosporium tenuissimum DF11, successively follows the steps below:
1), select that size is suitable, healthy growth plantation is in indoor Radix Salviae Miltiorrhizae plant, interception is about 5cm Radix Salviae Miltiorrhizae root and to the greatest extent
The soil for being attached to root surface may be removed clean.
2) it is used aseptic water washing 2 times again after, the PBS solution that the Radix Salviae Miltiorrhizae root segment of interception sterilizes is rinsed 2 times.It then will be red
Ginseng root segment is immersed in the sterilized PBS solution of 200mL, ultrasonic vibration 10min.
3) red sage root, is taken out, aseptic water washing 5 times, flushing liquor and the PBS solution after previous step ultrasonic vibration are uniformly mixed
It closes, suspension is made.
4) it, takes 500 μ L uniform suspensions to be coated on PDA plate, after 28 DEG C of cultures for 24 hours, carries out sterile verifying.
5) it, is such as generated without bacterium colony, then continues subsequent step 6)；Generated if any bacterium colony, then return step 2) continue root
Processing, so that it is guaranteed that surface sterilization is thorough.
6), suspension is cultivated 5-7 days in improvement martin substratum in 28 DEG C；
The strain culture of acquisition is inoculated in Radix Salviae Miltiorrhizae aseptic seedling root to carry out promoting the synthesis of Radix Salviae Miltiorrhizae root system effective component
Experiment, selects the optimal progress following step 7 of synthetic effect)；
7), the optimal strain culture of the resulting promotion Radix Salviae Miltiorrhizae root system effective component compound experiment effect of step 6) is carried out
It isolates and purifies, to obtain single bacterium kind, by single bacterium kind in same medium (improvement martin substratum) scribing line purifies and separates, directly
To acquisition pure culture.
This single colonie after purification is subjected to ITS sequencing and determines bacterial strain, and preservation through sequence alignment, obtains superfine branch spore
Mould (Cladosporium tenuissimum) DF11.
The Radix Salviae Miltiorrhizae endogenetic fungus plate and microscopic features are as follows:
Colony diameter about 20mm, surface villiform have deeper gauffer, are laid in media surface, blackish green.Colony edge
Mycelia growth is neat, and the back side is cracked in star.Mycelia branches separate, conidiophore are in chain, have branch in dendroid, easily
It is disconnected.
The rRNA result of gene sequence determination of Radix Salviae Miltiorrhizae endogenetic fungus of the present invention, ITS sequence segment are as shown in Figure 5.
Sequencing result is subjected to sequence alignment (https: //blast.ncbi.nlm.nih.gov/Blast.cgi) in the website NCBI, with
The similitude of superfine mould (Cladosporium tenuissimum) XCHK1 of branch spore is 99.81%.
The preservation information of the bacterial strain is as follows:
Preservation title: the superfine mould Cladosporium tenuissimum of branch spore；Depositary institution: Chinese microorganism strain is protected
Hide administration committee's common micro-organisms center, preservation address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3；Deposit number:
CGMCCNo.16883, preservation date 2018.11.26.
The culture of embodiment 2, Radix Salviae Miltiorrhizae aseptic seedling, successively follows the steps below:
1) the full seed of Salvia miltiorrhiza Growth, is chosen, is rinsed through flowing water, 75% ethanol postincubation 1min, rinsed with sterile water, 5%
After NaClO solution is handled 1~2 minute, rinsed with sterile water 2~3 times, excessive moisture is sucked with aseptic filter paper, is seeded in MS culture
In base, Radix Salviae Miltiorrhizae aseptic seedling is obtained.
2), proliferation seedling is cultivated:
After the long budding of aseptic seedling, in the band blade stem section merging MS culture medium of 1.5~2.0cm of clip in 25 DEG C of tissue culture rooms into
Row is cultivated, until grow up to the seedling of 3-4 piece leaf, is then transplanted to equipped with 100mlMS fluid nutrient medium and appropriate sterile
In the 1300ml PVC plastic tissue culture bottle of matrix (fertile soil: perlite: vermiculite=3:1:1 volume ratio mixture), 25 DEG C of groups
It trains room to cultivate 20~30 days, obtains aseptic seedling system.
Embodiment 3: influence of the Radix Salviae Miltiorrhizae endogenetic fungus to Radix Salviae Miltiorrhizae plant effective component biosynthesis:
Radix Salviae Miltiorrhizae aseptic seedling of the present invention and bacterial strain DF11 are co-cultured, successively followed the steps below:
(a) Radix Salviae Miltiorrhizae endogenetic fungus DF11 strain of the invention is taken, aseptically, with a small amount of mycelia of transfer needle picking,
Access in sterilized potato glucose liquid (PDA culture medium) fermentation flask, in 28 DEG C activation culture 6-7 days；Institute will be cultivated
It obtains liquid to be diluted, obtains concentration 106The bacteria suspension of CFU/ml or so.
(b) in the place inoculation bacteria suspension 1mL (concentration 10 of Radix Salviae Miltiorrhizae aseptic seedling root contacting substrate6CFU/ml or so)；It is red
Joining the potting soil matrix that endogenetic fungus and Radix Salviae Miltiorrhizae co-culture is fertile soil: perlite: the mixing of vermiculite=3:1:l (volume ratio) is planted
Train matrix；Holder: 1300ml PVC plastic tissue culture bottle；Hot-house culture temperature: 25 DEG C；In this, as experimental group (DF11).
Bacteria suspension is replaced with the PDA liquid medium of 1mL, remaining is equal to step (b), and gained processing is known as control group
(CK)。
Illustrate: experimental group (DF11), experimental group (DF11) starting " Radix Salviae Miltiorrhizae aseptic seedling " used are consistent, that is, without conspicuousness
Difference.
Plant strain growth situation is observed after 60 days, Radix Salviae Miltiorrhizae root is compared with the obvious thickening of control group after experimental group processing, and main root becomes
It is red.
The measurement of active constituent content in experiment 1, Radix Salviae Miltiorrhizae plant root:
Embodiment 3 is cultivated into 60 days resulting red sage roots in 45-50 DEG C of oven dried up to constant weight, is then ground to powder,
Extract (20mg red sage root powder is dissolved in 70% methanol of 2ml (V/V)) with methanol, handle 45min under ultrasound, 10000rpm from
Heart 15min, takes 0.22 μm of membrane filtration of supernatant, and extracting solution application high performance liquid chromatography (HPLC) system carries out tanshinone
With the analysis of danshinolic acid effective constituents.
High performance liquid chromatography (HPLC) condition are as follows: equipment: Waters e2695 binary high performance liquid chromatograph, detection
Device: 2996 diode array detector of Waters, chromatographic column: Waters SunFire C18 (250mm × 4.6mm, 5 μm), number
According to acquisition software: Empower 3.Chromatographic condition: flow velocity 1ml/min, 30 DEG C of column temperature, 10 μ l of loading volume.Detection wavelength: Radix Salviae Miltiorrhizae
Ketones component 270nm and liposoluble ingredient 288nm, mobile phase are respectively 0.026% phosphate aqueous solution and acetonitrile, gradient elution journey
Sequence is as follows:
The taproot and lateral root quantity increase that can be seen that processing group in conjunction with data in Fig. 2 and Fig. 3 is thicker, and color reddens, can
See that Radix Salviae Miltiorrhizae endogenetic fungus DF11 can promote Radix Salviae Miltiorrhizae root system development.Radix Salviae Miltiorrhizae endogenetic fungus DF11 is to effective component in Radix Salviae Miltiorrhizae plant
Content influences more significant (Fig. 4), Cryptotanshinone, dihydrotanshinone I, Tanshinone I, tanshinone IIA, and tanshin polyphenolic acid B is respectively pair
According to 35.53 times of group, 9.63 times, 8.98 times, 25.49 times and 1.71 times, prompt DF11 that can effectively facilitate effective component in Radix Salviae Miltiorrhizae
Biosynthesis.
" the branch spore (Cladosporium sp.) of CGMCC No.6608 " is substituted DF11 of the invention by comparative example 1, is pressed
It is detected according to above-described embodiment 3 and 1 the method for experiment, Cryptotanshinone only improves only 5~8 times relative to control group；Far
Far below DF11 of the invention.
Comparative example 2, other bacterial strains screened during invention, Cryptotanshinone only improve only about relative to control group
10 times；Well below DF11 of the invention.
The above list is only a few specific embodiments of the present invention for finally, it should also be noted that.Obviously, this hair
Bright to be not limited to above embodiments, acceptable there are many deformations.Those skilled in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Sequence table
<110>Institutes Of Technology Of Zhejiang
<120>superfine branch spore is mould and its promotes the purposes of Radix Salviae Miltiorrhizae root system effective component synthesis
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 541
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gcctgcggag ggatcattac aagagacacc ggtctaacca ccgggatgtt cataaccctt 60
tgttgtccga ctctgttgcc tccggggcga ccctgccttc gggcgggggc tccgggtgga 120
cacttcaaac tcttgcgtaa ctttgcagtc tgagtaaact taattaataa attaaaactt 180
ttaacaacgg atctcttggt tctggcatcg atgaagaacg cagcgaaatg cgataagtaa 240
tgtgaattgc agaattcagt gaatcatcga atctttgaac gcacattgcg ccccctggta 300
ttccgggggg catgcctgtt cgagcgtcat ttcaccactc aagcctcgct tggtattggg 360
caacgcggtc cgccgcgtgc ctcaaatcga ccggctgggt cttctgtccc ctaagcgttg 420
tggaaactat tcgctaaagg gtgctcggga ggctacgccg taaaacaaac ccatttctaa 480
ggttgacctc ggatcaggta ggcacaccag ctgaacttaa gcatatcaaa aggcggagga 540
a 541
Claims (5)
1. a kind of Radix Salviae Miltiorrhizae endogenetic fungus DF11, it is characterised in that: be the mould Cladosporium tenuissimum of superfine branch spore, protect
Hiding number is CGMCC No.16883.
2. the purposes of Radix Salviae Miltiorrhizae endogenetic fungus DF11 as described in claim 1, it is characterized in that: promoting Radix Salviae Miltiorrhizae root system effective component
Synthesis, the growth for promoting Radix Salviae Miltiorrhizae root system.
3. the purposes of Radix Salviae Miltiorrhizae endogenetic fungus DF11 described in requiring 2 according to right, it is characterized in that: the effective component is pellet
Join letones, danshinolic acid substance；
The tanshinone substance includes: Cryptotanshinone, dihydrotanshinone, Tanshinone I, tanshinone IIA,
The danshinolic acid substance includes Rosmarinic acid, tanshin polyphenolic acid B.
4. the method for improving active constituent content in Radix Salviae Miltiorrhizae, it is characterized in that: it is sterile that Radix Salviae Miltiorrhizae endogenetic fungus DF11 is seeded to Radix Salviae Miltiorrhizae
Seedling root co-cultures Radix Salviae Miltiorrhizae aseptic seedling and Radix Salviae Miltiorrhizae endogenetic fungus DF11 in (25 ± 0.5) DEG C；Incubation time is (60 ± 5) day.
5. the method according to claim 4 for improving active constituent content in Radix Salviae Miltiorrhizae, it is characterized in that: sterile in each Radix Salviae Miltiorrhizae
The bacteria suspension 1mL of Radix Salviae Miltiorrhizae endogenetic fungus DF11 is inoculated at the contacting substrate of seedling root；
The concentration of bacteria suspension is 106CFU/mL。
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