CN104877943B - One plant preventing and treating Radix rehmanniae root rot Antagonistic Fungi and its application - Google Patents
One plant preventing and treating Radix rehmanniae root rot Antagonistic Fungi and its application Download PDFInfo
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Abstract
The present invention provides the antagonistic bacterium of one plant of preventing and treating Radix rehmanniae root rot, and the bacterium is identified as pseudomonas aeruginosa, is named asPseudomonas aeruginosaLK 12, preserving number is CGMCC 10966.Pseudomonas aeruginosa LK 12 disclosed by the invention is to screen to obtain from glutinous rehmannia continuous cropping rhizosphere soil through flat board opposite culture method, to separating to the Fusarium oxysporum of glutinous rehmannia diseased region, Aspergillosis Huang opportunistic pathogen has strong antagonism, simultaneously to separating to the Fusarium oxysporum of radix pseudostellariae site of pathological change, fusarium moniliforme, the pathogens such as ankle section mould also have obvious inhibitory action, the root rot disease of the medicinal plant including glutinous rehmannia can effectively be prevented and treated, strain is provided for the cultivating disease that overcomes or alleviated by medicinal plant, it is that a kind of biocontrol effect is good, it is pollution-free, the ecological biological control potentiality bacterial strain of safety, development prospect is wide.
Description
Technical field
The present invention relates to one plant of bacterium bacterial strain, the antagonistic bacterium of more particularly to one plant preventing and treating Radix rehmanniae root rot, available for gram
Clothes alleviate continuous cropping glutinous rehmannia soil-borne disease problem, belong to microorganism and technical field of biological control.
Background technology
Understanding for Soil-sickness Problem is long-standing, many cereal crops（Such as wheat, potato）, industrial crops
（Such as tobacco, cotton）, oil crops（Such as soybean, peanut）, vegetables（Cucumber, tomato）, garden crop（Such as watermelon, strawberry）, medicine
Use plant（Such as ginseng, glutinous rehmannia, pseudo-ginseng）And artificial forest（Such as willow, China fir）All there is different degrees of Soil-sickness Problem.
But, the Soil-sickness Problem of long-standing problem China agricultural production is showed in Chinese medicine cultivation production and is particularly acute, and about 70%
Block medicinal plants all there is different degrees of Soil-sickness Problem, such as glutinous rehmannia, pseudo-ginseng, Radix Angelicae Sinensis, ginseng medicinal plant
There is serious Soil-sickness Problem.
Glutinous rehmannia（Rehmannia glutinosaL.）For Scrophulariaceae herbaceos perennial, it is used as medicine with root tuber, its medication
With a long history, clinical efficacy is the famous genunie medicinal materials of China significantly.However, the Soil-sickness Problem of glutinous rehmannia is particularly acute, even
Make the symptom that lower glutinous rehmannia plant often shows retarded growth：Overground part plant it is short and small reduction, leaf photosynthesis weaken,
Easily withered lodging；Underground part root tuber can not normally expand, root/shoot ratio imbalance；Reduction in the life period, effective component can not be accumulated effectively,
Medicinal quality decline；Wildness occurs for pest and disease damage, and root tuber is perishable, the normal withered phenomenon of occurrence of large-area（As shown in Figure 1）.Generally,
Must be spaced after Huang harvest per stub land can again plant for 8~10 years on same plot.Glutinous rehmannia continuous cropping not only causes yield, product
Matter drastically declines and soil-borne disease is serious, more it is a concern that being still not clear in the continuous cropping obstacle origin cause of formation and the mechanism of action
In the case of, most of peasant household attempts to maintain yield by increasing the measures such as fertilizer, pesticide abuse, but effect is often not
It is good, Productive statistics are not only increased, also result in that environmental pollution, Chinese medicine agriculture be residual exceeded and farmland ecosystem functional deterioration etc. one
Series of problems, produces the cultivation of natural resources of Chinese medicinal materials and has been absorbed in vicious circle.Therefore, building a kind of reasonable effective measures is used for gram
Clothes alleviate the important content of continuous cropping obstacle and soil-borne disease problem as resources of medicinal plant ecological study.Seminar's early stage
Research discovery, Fusarium oxysporum（As shown in Figure 2）It is the key factor for causing glutinous rehmannia continuous cropping obstacle, soil-borne disease rampant, it contains
Amount is made time limit increase with henry munronia herb and is continuously increased.And the biological control for carrying out plant disease using Antagonistic Fungi is considered as a kind of
Science, reasonable, efficient, environmentally friendly measure.The present invention is directed to glutinous rehmannia specialized form Fusarium oxysporum, and separation screening is to one plant
The antagonistic bacterium of the efficient antagonism disease fungus, is accredited as pseudomonas aeruginosa, is named asPseudomonas aeruginosa
LK-12。
The content of the invention
It is an object of the invention to provide the antagonistic bacterium of one plant of preventing and treating Radix rehmanniae root rot, continuously planted for biological control glutinous rehmannia
Common root rot problem during plant, biocontrol bacterial strain is provided to overcome or alleviated by glutinous rehmannia Soil-sickness Problem.
Technical scheme is as follows：
Antagonistic bacterium provided by the present invention is pseudomonas aeruginosa（Pseudomonas aeruginosaLK-12）, should
The Institute of Microorganism, Academia Sinica that bacterial strain has been preserved in Yard 1, BeiChen xi Road, Chaoyang District, Beijing City on June 9th, 2015
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC 10966.
Pseudomonas aeruginosa of the present invention（Pseudomonas aeruginosaLK-12）To glutinous rehmannia specialized form disease
Opportunistic pathogen-Fusarium oxysporum has very strong antagonism.
Beneficial Antagonistic Fungi of the present invention has following advantage：
（1）Antagonistic Fungi-pseudomonas aeruginosa of the present invention（Pseudomonas aeruginosaLK-12）It is logical
Cross what substantial amounts of screening operation was screened, screen and obtain from 2946 plants of soil bacterias, to the sharp spore reaping hook of glutinous rehmannia specialized form
Bacterium has very strong antagonistic effect.
（2）Antagonistic Fungi-pseudomonas aeruginosa of the present invention（Pseudomonas aeruginosaLK-12）To sieve
Select in glutinous rehmannia lesion root aspergillus flavus and screen Fusarium oxysporum in medicinal plant radix pseudostellariae continuous cropping soil or diseased region,
Naked section mould, beading mould also have very strong antagonism.
（3）Antagonistic Fungi-pseudomonas aeruginosa of the present invention（Pseudomonas aeruginosaLK-12）Source
In continuous cropping glutinous rehmannia rhizosphere soil, be not originate from other habitats, rhizosphere colonization effect is preferable and safe and reliable, to glutinous rehmannia and its
He often plants crop（Such as wheat, corn, paddy rice, soybean, peanut）Without the infection ability that causes a disease.
（4）Antagonistic Fungi-pseudomonas aeruginosa of the present invention（Pseudomonas aeruginosaLK-12）Training
Foster temperature range is preferably 25 °C~45 °C, and pH scopes are 5~9.5, and the temperature and pH scopes for being adapted to growth are wider, adaptability
By force.
Brief description of the drawings
Fig. 1 is the glutinous rehmannia overground part and underground part growing state of the different continuous cropping time limits.
Fig. 2 is the colonial morphology A for pathogen-Fusarium oxysporum that glutinous rehmannia plant diseases root is separated to：Represent bacterium colony
Front；B：Represent bacterium colony reverse side.
Fig. 3 is Antagonistic Fungi pseudomonas aeruginosa（Pseudomonas aeruginosaLK-12）Gram's staining and aobvious
Titanium miniplate is observed.
Fig. 4 is pseudomonas aeruginosa（Pseudomonas aeruginosaLK-12）Shone with the opposite culture of disease fungus
Piece plate centers are inoculated with all kinds of disease funguses, wherein A：Fusarium oxysporum（Source glutinous rehmannia）；B：Aspergillus flavus（Source glutinous rehmannia）；C：
Fusarium oxysporum（Source radix pseudostellariae）；D：Ankle section mould（Source radix pseudostellariae）；E：Fusarium moniliforme（Source radix pseudostellariae）B is horse
Bell potato sucrose culture medium（PSA）Flat board, remaining is potato glucose（PDA）Culture medium.
Fig. 5 is pseudomonas aeruginosa（Pseudomonas aeruginosaLK-12）Metabolite suppress sharp spore reaping hook
Bacteria growing A：Add LK-12 zymotic fluids；B：It is not added with LK-12 zymotic fluids.
Fig. 6 is pseudomonas aeruginosa（Pseudomonas aeruginosaLK-12）Prevention and control Fusarium oxysporum infects glutinous rehmannia
Tissue-cultured seedling A：Expression is only inoculated with Fusarium oxysporum（It is left）And inoculation Fusarium oxysporum and pseudomonad（It is right）Tissue-cultured seedling growth
Situation top view；B：Expression is only inoculated with Fusarium oxysporum（It is left）And inoculation Fusarium oxysporum and pseudomonad（It is right）Tissue-cultured seedling life
Long situation side view.
Fig. 7 is pseudomonas aeruginosa（Pseudomonas aeruginosaLK-12）Prevention and control Fusarium oxysporum infects glutinous rehmannia
Three basins of a detoxification transplanted seedling left sides represent only to be inoculated with Fusarium oxysporum；Right three basins represent to be inoculated with pseudomonad（Away from the cm of root 2 inoculations
One circle）And Fusarium oxysporum（Enclosed away from the cm of root 4 inoculations one）.
Embodiment
Technical scheme is described in detail below in conjunction with specific embodiment.Following examples are used only for
The description and interpretation present invention, without constituting the limitation to technical solution of the present invention.
The Antagonistic Fungi pseudomonas aeruginosa of embodiment 1（Pseudomonas aeruginosaLK-12）Screening and its mirror
It is fixed
1st, glutinous rehmannia specialized form Fusarium oxysporum Antagonistic FungiPseudomonas aeruginosaLK-12 screening
Antagonistic Fungi pseudomonas aeruginosa of the present invention（Pseudomonas aeruginosaLK-12）From continuous cropping
Screening is obtained in yellow rhizosphere soil.
1.1 pseudomonad selective mediums are prepared
Pseudomonad selective medium（Pseudomonas selective isolation agar, PSIA）Preparation side
Method is as follows：Weigh 20 g Soybean-casein digest agar mediums（Soybean casein digest agar, SCD）
（BD, USA）, plus 495.5 mL distilled water heating stirrings, fully after dissolving, add 1 mL 0.1%（wt/vol）Crystal violet is stored up
When being cooled to about 50 °C, 3.5 mL 5% are added into culture medium again by standby liquid, 121 °C of min of autoclave sterilization 15（wt/
vol）Nitrofurantoin storing solution, after fully mixing, pseudomonad selective medium is made after cooled and solidified and puts down for rapid a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices
Plate.
Wherein, 0.1%（wt/vol）The compound method of crystal violet storing solution is：Weigh 0.1 g crystal violets（Sangon, on
Sea）, it is dissolved in 100 mL distilled waters, room temperature preservation is standby；The compound method of Nitrofurantoin storing solution is：Weigh 5 g furans appropriate
Cause（Sangon, Shanghai）, it is added to 90 mL DMFs（Sangon, Shanghai）In fully dissolve, then be settled to
100 mL, room temperature is kept in dark place standby.
Pseudomonad separation screening in 1.2 rhizosphere soils
5 g fresh rhizosphere soil is weighed, in the sterilized water for being dissolved in 45 mL coolings, fully vibration shakes up, and obtains 10-1Dilution
Liquid, takes 5 mL 10-1In the sterilized water that the Soil Slurry of dilution factor is cooled down to another mL of bottle 45, fully vibration shakes up, and obtains 10-2Dilution, dilute again 10-3Dilution, draws 60 μ L soil dilution liquid, even spread to the pseudomonad choosing made
On selecting property culture medium flat plate, each dilution factor applies 3 flat boards, is placed in the h of lucifuge culture 30 in 32 °C of constant incubators.Select
Single bacterium colony is clear and legible and grows uniform flat board, is forwarded to 32 °C of continuation on new selective medium flat board and cultivates, treats bacterium
When falling high-visible, of short duration in 4 °C of refrigerators save backup is placed in.
The Antagonistic Fungi screening of 1.3 glutinous rehmannia specialized form Fusarium oxysporums
Prepare potato sucrose culture medium（PSA）Or potato dextrose medium（PDA）, it is formulated as follows：Weigh clean
The g of potato 200 of peeling, is cut into small pieces, plus suitable quantity of water boils the rear min of timing 25, with four layers of filtered through gauze, adds into filtrate
Enter the g of sucrose 30（Or the g of glucose 30）, the g of agar 15, continue heating stirring and mix, add water after slightly cooling down and be settled to 1000
ML, packing a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices or is saved backup after autoclaving.
Flat board is made after PSA or PDA culture medium through 121 °C of autoclave sterilizations, rapid a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, cooled and solidified, in training
Support ware bottom and cross the center of circle and cross frame, in the pseudomonad of the activated culture of inoculation apart from the cm of the center of circle 2.5 at, be placed in 28 °C of perseverances
In warm incubator after the h of lucifuge culture 48, glutinous rehmannia specialized form Fusarium oxysporum is inoculated with the center of culture dish, 28 °C are placed in
Lucifuge opposite culture a couple of days in constant incubator, the presence or absence of Real Time Observation inhibition zone and size.After opposite culture 5 days, by seeing
Examine inhibition zone size and filter out one plant of pseudomonad to glutinous rehmannia specialized form Fusarium oxysporum with strong antagonistic effect, to the bacterial strain
Preservation is carried out, deposit number is CGMCC 10966.The strong antagonistic strain screened is subjected to purifying culture, and inclined-plane is preserved
It is standby.
2nd, the antagonism dientification of bacteria of glutinous rehmannia specialized form Fusarium oxysporum
2.1 Antagonistic Fungis of the present invention（Deposit number is：CGMCC 10966）Through microexamination it can be seen that the bacterium is
Bacillus, the single flagellum of tool, Gram-negative bacteria（As shown in Figure 3）.It is bacterium colony that is flat, moistening, bacterium colony on LB solid mediums
Metal luster.Fluorchrome can be produced.Meanwhile, the physio-biochemical characteristics of the Antagonistic Fungi are：Oxidase positive, can oxidation Decomposition
Glucose, the sour not aerogenesis of production, while fructose, glycerine, mannitol, proline, arginine, alanine can be decomposed, but is not decomposed
Sucrose and inositol.Hydrolysis starch is unable to, can liquefy gelatin, decomposable asymmetric choice net urea, ammonium sulfate, ammonium chloride, ammonium nitrate, potassium nitrate.
2.2 16s-23s rRNA gene intervals PCR are identified
By the strong antagonistic strain filtered out（Deposit number is：CGMCC 10966）LB fluid nutrient mediums are forwarded to be expanded
Big culture, extracts genomic DNA, PCR amplification 16s-23s rRNA gene intervals, for bacteria molecule identification.Bacterial genomes
DNA extraction method is as follows：1 mL is taken to shake pseudomonad bacterium solution overnight（28 °C, 180 rpm）, 10000 rpm, 5 min of centrifugation,
Supernatant is removed, 950 μ L TE buffer solutions suspension precipitation is added into precipitation thalline, and add the SDS solution of 50 μ L 10% and 5 μ L eggs
White enzyme K（20mg/mL）, mix, 37 °C of h of water-bath 1 add the mol/mL NaCl solutions of 150 μ L 5 and 150 μ L CTAB/
NaCl solution（10% CTAB, 4.1% NaCl）, mix, 65 °C of min of water-bath 20, plus isometric Fen ︰ Lv Fang ︰ isoamyl alcohol（Body
The product ratio ︰ 1 of 25 ︰ 24）Solution is stripped, and 12000 rpm centrifuge 10 min, by supernatant with isometric chloroform/isoamyl alcohol
（24/1）Extract again once, supernatant is centrifuged 10 min, abandoned with isometric min of isopropanol precipitation at room temperature 30,12000 rpm
Clearly, DNA precipitations are cleaned with 70% ethanol, are dissolved after natural air drying with 50 μ L sterilized waters.
Molecular Identification is carried out to the pseudomonad of separation screening using 16s-23s rRNA gene interval PCR amplification techniques,
Identify that primer sequence is：1405f（5'-TGYACACACCGCCCGT-3'）And 456r（5'-CCTTT CCCTCACGGTACTG -
3'）.PCR amplification system（25 μL）For：12.5 μL Taq PCR Master Mix (2×)（Sangon, Shanghai）, 1.0 μ L
Upstream and downstream primer（10 μM）, 20 ng DNA profilings.PCR amplification programs are：94 °C of pre-degenerations 5 min, 94 °C of 1 min of denaturation,
61 °C of 1 min of annealing, 72 °C of 90 sec of extension, 35 circulations continue 72 °C of 10 min of extension.The 16s-23s rRNA of amplification
Genetic fragment detects with 1% agarose gel electrophoresis, and with Universal DNA Purification Kit glue reclaim kits
（TIANGEN, Beijing）Purify and reclaim purpose band, and serve the raw work sequencing portion in sea and be sequenced.Sequencing sequence uses BLAST works
Tool and ncbi database（Nucleotide collection（nr/nt））It is compared.The molecular biology identification antagonism
Bacterium is pseudomonas aeruginosa, is named asPseudomonas aeruginosa LK-12。
The fungistatic effect detection of the Antagonistic Fungi of embodiment 2 and its antagonistic substance
（1）Antagonistic Fungi opposite culture
Prepare PSA or PDA culture medium, after 121 °C of autoclave sterilizations after rapid a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices, cooled and solidified obtained PSA or
PDA plate, the center of circle is crossed in culture dish bottom and is crossed frame, and the false unit cell of activated culture is being inoculated with the cm of the center of circle 2.5
Bacterium, is placed in 28 °C of constant incubators after the h of lucifuge culture 48, is inoculated with disease fungus in the center of culture dish, is placed in 28 °C
Lucifuge opposite culture a couple of days in constant incubator, the formation of Real Time Observation inhibition zone.Culture discovery in 5 days, pseudomonas aeruginosa
（Pseudomonas aeruginosaLK-12）Being capable of efficiently antagonism glutinous rehmannia specialized form Fusarium oxysporum, aspergillus flavus and crown prince
Join specialized form Fusarium oxysporum, fusarium moniliforme, ankle section mould, suppress its mycelial growth（As shown in Figure 4）.
（2）The fungistatic effect detection of antagonistic substance
The Antagonistic Fungi of preservation is inoculated in LB fluid nutrient mediums after shaken cultivation 48h, high speed centrifugation, 0.22 μ of supernatant
M filtering with microporous membrane is degerming, and filtrate is carried out into fungistatic effect evaluation using metabolin flat board additive process.As a result show, with the addition of
The test group of metabolite, pathogen Fusarium oxysporum mycelial growth is suppressed, and is not added with the control group of metabolite, cause of disease
Bacterium mycelial growth is normal.It is small by the mycelia radius ratio control group for measuring the test group Fusarium oxysporum for finding addition metabolite
1 cm（As shown in Figure 5）.
The experimental procedure of wherein metabolin flat board additive process is：Sterilized PDA culture medium is heated to complete thawing, it is cold
But to 45 DEG C or so the antagonism fermented liquids for adding heat sterilization（2.5%）, poured into after mixing in culture dish, treat that it solidifies.It
The glutinous rehmannia specialized form Fusarium oxysporum activated is accessed afterwards, is placed in lucifuge culture a couple of days, Real Time Observation in 28 °C of constant incubators
Fusarium oxysporum grows.
The Antagonistic Fungi prevention and control Fusarium oxysporum of embodiment 3 encroaches on glutinous rehmannia tissue-cultured seedling effect assessment
With prepared glutinous rehmannia tissue-cultured seedling MS culture mediums（The g/L sugarcanes of+0.2 mg/L IBA of the mg/L of MS+0.2 6-BA+30
Sugared+7 g/L agar）, each tissue culture bottle adds 30 mL culture mediums, through autoclave sterilization, after solidification to be cooled, uses tweezers
A ditch is burnt in media surface, 2 plants of inoculation glutinous rehmannia seedling, is placed in 25 °C of constant temperature tissue culture rooms and cultivates 45 in ditch side
After it, the μ L of pseudomonad LK-12 bacterium solutions 300 of overnight incubation, control group addition equivalent LB culture medium generations are added in ditch
Replace, in the opposite side of ditch（I.e. away from glutinous rehmannia tissue-cultured seedling side）Glutinous rehmannia specialized form Fusarium oxysporum is inoculated with, is during which continued toward small
Addition pseudomonad LK-12 bacterium solutions in ditch, dynamic observation Fusarium oxysporum grows and its infects situation to glutinous rehmannia tissue-cultured seedling.Knot
Fruit is shown, between pathogen and tissue-cultured seedling in the test group added with Antagonistic Fungi, and Fusarium oxysporum growth is suppressed, only outside ditch
Grow, and added in the control group of equivalent LB culture mediums in the range of side, Fusarium oxysporum growth is quick, can cross ditch, invade
Glutinous rehmannia tissue-cultured seedling plant is contaminated, causes tissue-cultured seedling stem rot rotten, it is final dead（As shown in Figure 6）.
The Antagonistic Fungi prevention and control Fusarium oxysporum of embodiment 4 encroaches on glutinous rehmannia detoxification transplanted seedling effect assessment
By the glutinous rehmannia tissue culture transplantation of seedlings for cultivating 45 days into the culture matrix handled through autoclave sterilization, 1 is planted per basin
Strain, domestication culture two weeks selects the consistent glutinous rehmannia transplanted seedling of growing way and carries out follow-up test.2 cm around glutinous rehmannia transplanted seedling root
The pseudomonad LK-12 bacterium solutions of overnight incubation are enclosed in place's inoculation one, after 2 days, around root at 4 cm（That is pseudomonad LK-12
Outer ring）It is inoculated with glutinous rehmannia specialized form Fusarium oxysporum, 25 °C of constant temperature tissue culture rooms and continues to cultivate, addition in during which every 3 days is once false single
Born of the same parents' bacterium LK-12 bacterium solutions, control group is to add the replacement of equivalent LB culture mediums, and dynamic observation Fusarium oxysporum is invaded glutinous rehmannia transplanted seedling
Dye situation.As a result show：Only in the control group of addition pathogen, there are disease states quickly in glutinous rehmannia transplanted seedling, at the 10th day
Start wilt phenomenon occur, to death of being rotted substantially at 15 days, choosing bacterium, sequence verification finds control group soil surface and dead again
Dying on plant has a large amount of Fusarium oxysporum mycelial growths, and the glutinous rehmannia detoxic seedling well-grown of test group, during whole experiment all
There is no lesion phenomenon（As shown in Figure 7）.
As fully visible, one plant of Antagonistic Fungi that the present invention is filtered out（Preserving number is CGMCC 10966）To glutinous rehmannia specialized form disease
Opportunistic pathogen has very strong antagonism, can effectively infringement of the prevention and control Fusarium oxysporum pathogen to glutinous rehmannia, have in the field
Wide application prospect.
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, should all belong to the covering scope of the present invention.
SEQUENCE LISTING
<110>University Of Agriculture and Forestry In Fujian
<120>One plant preventing and treating Radix rehmanniae root rot Antagonistic Fungi and its application
<130> 2
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> DNA
<213>Artificial sequence
<400> 1
tgyacacacc gcccgt 16
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
cctttccctc acggtactg 19
Claims (2)
1. the Antagonistic Fungi of one plant of preventing and treating Radix rehmanniae root rot, it is characterised in that：Described Antagonistic Fungi is pseudomonas aeruginosa
（Pseudomonas aeruginosa）LK-12, is preserved in Chinese microorganism strain preservation management committee on June 9th, 2015
Member's meeting common micro-organisms center, culture presevation numbering is CGMCC 10966.
2. a kind of Antagonistic Fungi of preventing and treating Radix rehmanniae root rot as claimed in claim 1 is preventing and treating glutinous rehmannia, radix pseudostellariae medicinal plant again
Plant the application in disease and soil-borne disease.
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| CN110305796B (en) * | 2019-05-28 | 2020-06-12 | 山东省花生研究所 | Aspergillus flavus PAF-1 incapable of producing aflatoxin and application thereof |
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