CN109504611A - One plant of bletilla endogenetic fungus 1-G1 and its application - Google Patents

One plant of bletilla endogenetic fungus 1-G1 and its application Download PDF

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CN109504611A
CN109504611A CN201811588958.1A CN201811588958A CN109504611A CN 109504611 A CN109504611 A CN 109504611A CN 201811588958 A CN201811588958 A CN 201811588958A CN 109504611 A CN109504611 A CN 109504611A
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bletilla
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endogenetic fungus
bacterial strain
growth
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CN109504611B (en
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李良波
田佩雯
黄荣韶
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Guangxi University
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Abstract

The invention belongs to technical field of microbe application more particularly to one plant of bletilla endogenetic fungus 1-G1 and its applications.One plant of bletilla endogenetic fungus 1-G1, classification naming be between seat shell category (Diaporthe spectabilis), be preserved in Guangdong Province's Culture Collection on 06 06th, 2018, deposit number is GDMCC NO.60382.Separation screens one plant of endogenetic fungus (Diaporthe spectabilis) 1-G1 to the present invention inside medicinal plant bletilla for the first time, the bacterial strain has stronger growth-promoting functions to the growth of bletilla various aspects, brings wide application prospect for the yield and quality control of bletilla.

Description

One plant of bletilla endogenetic fungus 1-G1 and its application
Technical field
The invention belongs to technical field of microbe application more particularly to one plant of bletilla endogenetic fungus 1-G1 and its applications.
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.
Bletilla Bletilla striata (Thunb.) Reichb.F. is orchid family bletilla category herbaceos perennial, and name is white Chicken, horn seven, army ask son, Pseudobulbus Bletillae (Rhizoma Bletillae) etc., and bletilla is traditional one of the Chinese medicine in China, block that is fleshy hypertrophy and having stickiness Stem can be used as medicine, and have hemostasis, protection mucous membrane, antitumor, antibacterial and other effects, and few plant polyoses have effects that convergence, because The title that this is known as " must be puckery and receive, enter lung hemostasis, myogenic controls sore, and surgery is most kind " is bletilla syrup, bletilla electuary, stomach Kang Ling, fast The main prescription raw material of the Chinese patent drugs such as stomach piece.In recent years, the destruction with natural habitat is developed by degree due to wild bletilla, Wild bletilla resource straight line decline, the yield and quality of Bletilla striata medicinal materials cannot be guaranteed, and be classified as focused protection by country One of Wild Medicinal, it is in imminent danger at present.Since bletilla seed is without endosperm, it is difficult to be broadcast live seedling, and tissue-cultured seedling plantation at Motility rate is lower.It is difficult that this all allows to carry out bletilla large-scale plantation.
Currently, mostly use wild bletilla piecemeal stem to plant during the artificial cultivation of Guangxi province bletilla, seedlings plantation three Year harvests.But seedling quality standard lacks, kind and Quality Control Technology research and development application lag, and is all to restrict bletilla industry to hold The critical issue of supervention exhibition.Therefore, the endogenetic fungus that filtering out thus can promote bletilla to grow is of great significance, and screening is in turn Developing and using bletilla endogenetic fungus is also one of the measure that Bletilla striata medicinal materials quality is effectively ensured.
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
It is an object of the present invention to provide one plant of bletilla endogenetic fungus 1-G1, which has the function of promoting bletilla growth, from And bletilla growth can be effectively facilitated, improve yield, further improve the quality of bletilla.
Technical solution provided by the invention is as follows:
One plant of bletilla endogenetic fungus 1-G1, classification naming be between seat shell category (Diaporthe spectabilis), in 2018 It is preserved within 06 day 06 month Guangdong Province's Culture Collection year, deposit number is GDMCC NO.60382.
The present invention also provides the bletilla endogenetic fungus 1-G1 to promote the application in bletilla growth.
Compared with prior art, the invention has the following beneficial effects:
Separation screens one plant of endogenetic fungus (Diaporthe to the present invention inside medicinal plant bletilla for the first time Spectabilis) 1-G1, the bacterial strain have stronger growth-promoting functions to the growth of bletilla various aspects, are the yield and quality control of bletilla System brings wide application prospect.
Detailed description of the invention
Fig. 1 is bletilla endogenetic fungus 1-G1 colonial morphology figure of the invention；
Fig. 2 is the measurement of bletilla endogenetic fungus 1-G1 active bacterial strain fermentation liquid IAA content of the invention；
Fig. 3 is that bletilla endogenetic fungus 1-G1 active bacterial strain siderophores synthesis ability of the invention detects；
Fig. 4 is 24 plants of bletilla endogenetic fungus and its mutually fungi Phylogenetic Relationships.
Preservation information explanation
Diaporthe spectabilis, deposit number are GDMCC NO.60382, and the deposit date is 06 month 2018 06, depositary institution was Guangdong Province's Culture Collection (abbreviation GDMCC, address: GuangZhou, China city martyr Road 100 Numbers five building, Institute of Micro-biology, province laboratory building, postcode: 510070).
Specific embodiment
Following embodiment does not limit the present invention to better understand the present invention.Experimental method in following embodiments, It unless otherwise specified, is conventional method.The experimental materials used in the following example is unless otherwise specified conventional life Change what reagent shop was commercially available.
Quantitative experiment in following embodiments, is respectively provided with three repeated experiments, and results are averaged.
Embodiment 1: separation, screening and the identification of bacterial strain
The separation of 1.1 bacterial strains
Material to be tested: the wild bletilla of Guangxi Ziyuan County and Huan Jiang autonomous county is picked up from.
Culture medium: 1000ml potato dextrose agar (PDA culture medium)；PDA culture medium 300g containing potato, Glucose 20g, agar 20g, Medium's PH Value are 6.0 ± 0.2；DE culture medium CaCl containing 0.111mg2, 0.174mg K2SO4, 27.8mg FeSO4·7H20,6.501mg MnCl2·4H20,0.241mg NaMo4, 0.123mg MgSO4·7H20, 0.0544mg KH2PO4, 1.545mg H3BO4, 0.80516mg ZnSO4·7H20,52.108mg Na2EDTA·2H20,9g can Soluble starch, 1g yeast extract, 8g agar, 1000mL water, pH value 6.0.
Surface sterilization: by wild bletilla plant root and stem tuber soil and attachment gently shake off, rinsed under flowing water, It is spare that clean bletilla root and stem tuber are placed in suck dry moisture on filter paper.On superclean bench, bletilla root and stem tuber are placed in In sterile water, sufficiently oscillation is cleaned and is pulled out, carries out surface sterilization in order, surface sterilization 15s in 75% alcohol, 2% secondary chlorine Acid sodium solution sterilizes 6-10min, aseptic water washing 4-5 times.Using conventional tissue block partition method, after tissue surface is sterilized Bletilla root and stem tuber are placed on sterile filter paper, suck dry moisture.
The separation of bacterial strain, purifying: by after disinfection bletilla root and stem tuber be cut into the fritter of 1cm × 1cm, by root and stem tuber group It knits block to be inoculated in respectively on PDA plate culture medium, each culture dish is inoculated with 4 tissue blocks, is placed in mold incubator, 28 DEG C of perseverances Temperature, it is dark, it is inverted culture.To guarantee that bletilla root and stem tuber show sterilizing thoroughly, last time is taken to clean with sterile liquid-transfering gun The sterile water of bletilla root and stem tuber is coated in new PDA culture medium, is repeated 3 groups of culture dishes, is placed in mold incubator, 28 DEG C It is inverted culture 7d, if growing on PDA plate without miscellaneous bacteria, proves tissue surface disinfection thoroughly, isolated fungi is interior life Fungi.Bletilla stem tuber group is woven in PDA culture medium after culture 2-3d, can be observed have mycelia to grow from tissue block edge, to bacterium When filament length to macroscopic bacterium colony, with sterile toothpick from a small amount of mycelium inoculation of colony edge picking to new PDA culture medium On, repetition is forwarded to obtain pure bacterium colony.
The endogenetic fungus being separated to is inoculated in PDA plate, it is stand-by after being cultivated 7-10 days at 28 DEG C.
1.2 screening
The active primary dcreening operation of growth-promoting is carried out to for examination bletilla endogenetic fungus using bletilla aseptic seedling and endogenetic fungus symbiosis culture.
Symbiotic culture medium is configured in advance by DE culture medium prescription, in separating device 250mL tissue culture bottle, 121 DEG C every bottle, 30min It sterilizes spare.On superclean bench, the sterile tissue-cultured seedling of bletilla is taken out, the culture medium that root is taken out of is cleaned with sterile water, sets In suck dry moisture on aseptic filter paper, then move into DE culture medium.Every bottle connects 4 plants, arranges between two parties in " mouth " word, after the completion of switching It is put into culturing room and cultivates 15d, 25-26 DEG C of temperature, daily illumination 8-12h, intensity of illumination 2000-3000Lux.To bletilla without For vaccine after growing 15d on DE culture medium, growth conditions are basicly stable.At this point, respectively from the bletilla endogenetic fungus activated Female colony edge, which is beaten, takes bacteria cake that diameter is 0.6cm as inoculation material, picking pure culture biscuits involvng inoculation to the centre of 4 plants of bletilla seedlings, away from Seedling about 2cm, interior one piece of the inoculation of each tissue culture bottle, each bacterial strain repeat 3 bottles, and control group accesses sterile PDA agar block.It has been inoculated with At rear sealing, it is put in culturing room and cultivates, 25-26 DEG C of temperature, daily illumination 8-12h, intensity of illumination 2000-3000Lux.It is fixed Phase observes the growth conditions of fungi and bletilla tissue-cultured seedling.After symbiosis culture 30 days, with the bacterium speed of growth is moderate and bletilla aseptic seedling Normal growth is standard, and picking out can be with the fungi of the good symbiosis of bletilla aseptic seedling.The bacterial strain that primary dcreening operation is obtained, with identical side Method is reinoculated in DE culture medium, every bottle of 3 plants of aseptic seedlings, to access the sterile letheen block of same size as control.It is identical Condition of culture, after symbiosis culture 60d, take out control seedling and symbiosis seedling respectively, clean root remaining medium, blotted with filter paper Surface moisture.Measure its plant height, newborn radical, newly sprout number, stem tuber diameter.
As a result 5 plants are obtained to all very strong growth-promoting fungal bacterial strain of bletilla growth-promoting effect, wherein one plant of entitled 1-G1.
1.3 identification
(1) strain morphology feature
Morphological features observation: endophyte fungal bacterial strain 1-G1 to be identified is seeded in PDA culture medium, is placed in 28 DEG C culture, observed its cultural characteristic and color change at 5,14 and 20 days respectively.Take the color characteristic for stablizing maturation as its training Support feature, the foundation as identification.Observe and record the color of aerial hyphae, size, color, tissue profile, the surface shape of bacterium colony Shape etc. is used as fixed reference feature.
As shown in Figure 1, colony morphology characteristic: in PDA culture medium, bacterium colony is round, and culture substrate mycelium is in grey black, radiation Shape, aerial hyphae grey black, flocculence.
(2) bacterial strain ITS sequence and its phylogenetic analysis
The preparation of DNA profiling:
Reagent: (1) lysis buffer: Tris-Ac (pH 8.0) 20mM, 3%CTAB, 1.4mol/L NaCl, 10% DTT)；(2) saturated phenol: chloroform: isoamyl alcohol (25:24:1) mixed liquor, isopropanol, ddH2O；
DNA is extracted:
Take appropriate hypha,hyphae, be added 600 μ L CTAB Extraction buffers (20mmol/L Tris (pH=8.0), 3% CTAB, 1.4mol/L NaCl, 10%DTT), a little sterilizing quartz sand is added, mycelia is fully ground.Lapping liquid is moved into In the EP pipe of 1.5mL, during which 65 DEG C of water-bath 30min rock EP pipe once every 10min, mix lapping liquid.After 65 DEG C of water-baths 400 μ L saturated phenols: chloroform: isoamyl alcohol (25:24:1) mixed liquor are added in ice bath 10min, mix.12000rpm is centrifuged 10min. 400 μ L of supernatant is taken, the isopropanol of pre-cooling is added, after mixing, is stood, ice bath 1 hour, 12000rpm is centrifuged 10min, abandons supernatant Liquid dries, and is resuspended with the ddH2O of 20 μ L, then takes 2 μ L as pcr template PCR amplification ITS sequence.
(1) PCR instrument: ABI 2720-XL DNA sequencer (Applied Biosystems, USA)
(2) amplimer: ITS1 (5 '-T C C G T A G G T G A A C C T G C G G-3 ') and ITS4 (5′-T C C T C C G C T T A T T G A T A T G C-3′)
(3) amplification system: ITS sequence PCR amplification system is as shown in table 1.
1 ITS sequence PCR amplification system of table
Reactant Sample-adding amount
2X TagMasterMix 25μL
Primer-1 1μL
Primer-2 1μL
Template DNA 2μL
ddH2O 21μL
React total volume 50μL
PCR reaction condition is as shown in table 2.
2 PCR reaction condition of table
Note: step 2 carries out 30 circulations.
The electrophoresis detection of pcr amplification product:
Deposition condition be 1% Ago-Gel ((10000 × aqueous solution of GelRed containing Super, 1 μ L/10mL), 1 × TBE electrophoretic buffer, 90V electrophoresis 1 hour, PCR product applied sample amount was 4 μ L, point after mixing with 1 μ L Loading dye Sample.
Observation is as a result, using the DL1000DNA Marker of TaKaRa company as nucleic acid mark under gel imaging system ultraviolet lamp Quasi-molecule amount object of reference, determines expanding fragment length.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:
NCBI is logged in, the fungi rDNA ITS sequence that sequencing obtains is compared with the sequence in GenBank database, Its accession number, most like bacterial strain, similitude etc. are recorded, and downloads the rDNA ITS sequence of the bacterial strain, is gathered using MEGA 7 Alanysis and building NJ phylogenetic tree.The ITS sequence of 1-G1 is as shown in SEQ ID No.1.
The rDNA ITS sequence is subjected to Blast sequence analysis in GeneBank, obtains the highest representative of similitude Bacterial strain records its accession number, most like bacterial strain, similitude etc., and downloads the rDNA ITS sequence of the bacterial strain, building NJ system hair Educate tree (as shown in Figure 4).It is as shown in table 3 with the highest bacterial strain of bacterial strain 1-G1 similitude.
Table 3 and the nearest bacterial strain of fungi 1-G1 similitude
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.
The result shows that the fungi in bacterial strain 1-G1 and Diaporthe category has very high base sequence similitude, therefore download The reference strain sequence that Diaporthe belongs in GenBank, be used for Phylogenetic Analysis, is belonged to Diaporthe Diaporthe sp. (KX110392) is used as outer group.In the systematic evolution tree of building, 1-G1 and Diaporthe sp.'s is more A strain gets together to form the end branch that holding strength is 99%.
Base similarity system design the result shows that, 1-G1 and Diaporthe sp. base sequence do not have difference, sequence similarity It is 100%.Bacterial strain is initially identified as Diaporthe sp. by comprehensive morphological and molecular biological characteristics.
Embodiment 2: active bacterial strain growth-promoting Characteristics Detection
The detection of 2.1 active bacterial strain 1-G1 fermentation liquid heteroauxin (IAA) synthesis capabilities
1mg IAA is accurately weighed, the dissolution of 10mL distilled water is added to get the IAA reference substance solution of 100 μ g/mL.Gradient is dilute Releasing IAA reference substance solution is 10,20,30,40,50,60,70,80ug/mL.By IAA solution and the Salkowski ratio of each gradient 1:1 is mixed color liquid by volume, after room temperature avoid light place 30min, measures the OD530 of the IAA solution of each concentration.With IAA's Concentration is abscissa, and the IAA solution O D530 of each concentration is ordinate, draws standard curve.
20mL IAA is added in the centrifuge tube that 50mL can sterilize and detects culture medium, 121 DEG C of sterilizing 20min take out cooling It is spare.On superclean bench, the fungus block for taking 0.6cm is beaten at the edge of the female bacterium colony activated with punch, with sterile tooth Fungus block is inoculated into above-mentioned centrifuge tube by label respectively, and each test tube is inoculated with two pieces, is repeated 3 pipes, is inoculated with the sterile of same size Agar block is as blank control.28 DEG C of shaking table are placed in, 150r/min is cultivated 5 days.100 μ L culture solutions are taken to drip to whiteware plate On, 100 μ L Salkowski color solutions are added, by whiteware plate room temperature avoid light place 30min, observe color change, control The sterile culture medium of 100 μ L is only added in color solution, the IAA of 50mg/L and 25mg/L is added in positive control color solution.To have The strain cultured solution 8000r/min of obvious color change, is centrifuged 10min, 2mL supernatant is taken to cross 0.22 μm of miillpore filter respectively, After filtration sterilization, 2mL Salkowski color solution is added, after room temperature avoid light place 30min, detects culture medium with the IAA of blank Same treatment measures the OD530 of each mixed liquor as control zeroing, calculates sample referring to IAA standard curve obtained above IAA content, 3 repetitions, the unit of IAA content are μ g/mL.
As shown in Fig. 2, from left to right in positive control be respectively 100 μ g/mL and 25 μ g/mL IAA solution in 100 μ L are added Salkowski color solution, after 30min is placed in shading, mixed liquor color is pink whether there is or not discoloration, and IAA concentration is higher, color It is deeper.Fungi 1-N2 fermentation liquid is centrifuged, supernatant is taken to mix with Salkowski color solution, after 30min is placed in shading, mixing Liquid has apparent pink colour to change.
The fermentation liquid of fungi 1-G1 is taken to do IAA assay, as a result, IAA content is 42.38 μ g/mL.IAA is to growth Facilitation is chiefly to facilitate the elongation of the growth of cell, especially cell, can promote skewer rooting of cuttings.In vaccine symbiosis and In pot experiment, fungi 1-G1 shows apparent growth-promoting activity, and bacterial strain 1-G1 has the energy for relatively synthesizing IAA by force in the test Power, this result are consistent with the test result of fungi growth-promoting early period.
The detection of 2.2 bacterial strain synthesis capability siderophores
20mL iron-free czapek's medium is added in the centrifuge tube that 50mL can sterilize, 121 DEG C of sterilizing 20min take out cooling It is spare.On superclean bench, the fungus block for taking 0.6cm is beaten at the edge for having activated female bacterium colony with punch, fungus block is connect respectively Kind is into above-mentioned centrifuge tube, and each test tube is inoculated with two pieces, and each fungi repeats 3 pipes, and the sterile of same size is added in control Agar block, 28 DEG C, 150r/min is cultivated 5 days.Culture solution 8000rpm is centrifuged 10min, takes supernatant, on whiteware plate, 1:1 takes supernatant and CAS developing solution by volume, jiggles mixing, observes its color change, liquid color after record mixing Become red, the culture solution of purple or yellow, and photograph to record.It is red, the strain culturing of purple or yellow by color change Liquid is centrifuged 10min (8000rpm), and 2mL supernatant is taken to cross 0.22 μm of miillpore filter respectively, and after filtration sterilization, 2mL CAS ratio is added Color liquid is stored at room temperature after placing 1h, is returned to zero with distilled water, measure its OD680, be calculated as " As ", takes the iron-free Cha Shi training that 2mL is sterile Nutrient solution measures its OD680 as reference value by identical method, is calculated as " Ar ".It is determined by the change of the OD680 value of blank control The concentration of siderophore in culture solution.The concentration of siderophore is indicated with siderophore active unit (SU) in test, SU=[(Ar- As)/Ar] × 100%.SU value is bigger, indicates that synthesis siderophore ability is stronger, and siderophore is opposite in As/Ar expression culture solution Content, the value is smaller, shows that siderophore content is higher.Generally have high yield siderophore ability bacterium bacterium As/Ar less than 0.5.
In nature, ferro element exists mostly in the form of FeO and ferric hydroxide composite, this insoluble state biology It is difficult with.Siderophore is the small molecule chelate of metal ion generated by microorganism, to Fe3+With strong compatibility.
As shown in figure 3, fungi 1-G1 bacteria suspension takes on a red color with the mixing of CAS developing solution to react, sterile in 5 endogenetic fungus After culture solution is mixed with CAS, color is in light green color.Illustrate the ability that fungi 1-G1 has synthesis siderophore.
The quantitative determination of 4 bacterial strain of table production siderophore
As shown in table 4, the SU of bacterial strain 1-G1 is 0.5539, and As/Ar value illustrates the bacterial strain for high yield iron load less than 0.5 Body bacterial strain.
In conclusion bacterial strain bletilla endogenetic fungus 1-G1 has stronger production IAA ability, and there is the energy of synthesis siderophore Power.Also, apparent promotion growth is played in the test of seedling growth-promoting.Therefore, in terms of the ecologic planting of bletilla, bacterium Strain bletilla endogenetic fungus 1-G1 fermentation liquid has apparent potentiality.
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.
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Claims (2)

1. one plant of bletilla endogenetic fungus 1-G1, which is characterized in that classification naming be between seat shell category (Diaporthe Spectabilis), Guangdong Province's Culture Collection, deposit number GDMCC were preserved on 06 06th, 2018 NO.60382。
2. bletilla endogenetic fungus 1-G1 according to claim 1 is promoting the application in bletilla growth.
CN201811588958.1A 2018-12-25 2018-12-25 Bletilla striata endophytic fungus 1-G1 and application thereof Active CN109504611B (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113549558A (en) * 2021-09-06 2021-10-26 安徽农业大学 Novel tea tree endophytic biocontrol fungus Diaporthe australiana and application thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113549558A (en) * 2021-09-06 2021-10-26 安徽农业大学 Novel tea tree endophytic biocontrol fungus Diaporthe australiana and application thereof
CN113549558B (en) * 2021-09-06 2023-03-10 安徽农业大学 Novel tea tree endophytic biocontrol fungus Diaporthe australiana and application thereof

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