CN110396492A - One plant of walnut branch-rot antagonistic bacterium and its application - Google Patents
One plant of walnut branch-rot antagonistic bacterium and its application Download PDFInfo
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- CN110396492A CN110396492A CN201910822493.XA CN201910822493A CN110396492A CN 110396492 A CN110396492 A CN 110396492A CN 201910822493 A CN201910822493 A CN 201910822493A CN 110396492 A CN110396492 A CN 110396492A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
Abstract
The present invention relates to one plant of walnut branch-rot antagonistic bacterium and its applications, the bacterium is bacillus subtilis (Bacillus subtilis) QB002, deposit number is CGMCC NO.18218, present invention discover that QB002 is able to suppress the growth of walnut branch-rot bacterium mycelia, cause mycelia malformation and distortion, generate multiple branches, measuring it by plate opposite culture is 88.6% to the growth inhibition ratio of walnut branch-rot pathogen, and to canker of apple fruit, jujube fruit black spot, Lee's brown rot, there is certain inhibiting effect, there is broad-spectrum antibacterial ability；Bacillus subtilis QB002 provided by the invention has good Biocontrol Potential to walnut branch-rot, can be used for preventing and treating walnut branch-rot.
Description
Technical field
The invention belongs to microbiologies and plant disease prevention and control field, specifically, being related to a kind of pair of various plants cause of disease
Bacillus subtilis and its application with good inhibitory effect.
Background technique
Walnut (Juglans regia L.) is also known as English walnut, is the woody oil tree species of Juglandaceae juglans, is four big dry
First of fruit, nutritive value and medical value are very high.There are commerial growing, planting number in a provinces and cities, China more than 20 at present
It occupies first place in the world with yield.Xinjiang is one of source area and main producing region of walnut, and Xinjiang walnut yield has accounted for national total yield
The 15% of amount, Walnut Industry has become the important component of Xinjiang economy development.However by fungi Juglanconis
Walnut branch-rot caused by juglandina occurs seriously, to cause branch chlorisis, dry up, seriously affect in Xinjiang walnut producing region
The health of trees and the yield of walnut and quality.The germ can also endanger the blade and fruit of walnut simultaneously, on aggrieved blade
Subcircular scab is formed, anaphase blade surface occurs and is covered by scab, the photosynthesis of tree body is seriously affected；Walnut Fruit is aggrieved
Black scab is formed in fruit surface afterwards, influences the yield and quality (Fig. 1) of fruit.The general diseased plant rate 20% of walnut branch-rot
~30%, diseased plant rate reaches 50% or more when serious, causes a large amount of branches withered, directly affects the matter of Growth status and walnut
Amount, causes huge economic loss to orchard worker.
Currently, controlling disease by fungicide such as application Mancozebs in production to walnut branch-rot based on chemical prevention
Feelings sprawling not only breaks up the environmental health in orchard, and influences walnut quality, cause pesticide residue, soil micro-ecosystem unbalance etc.
Problem.Biological control then reduces the generation and extension of disease from ecological safety and protection environmental.Therefore, utmostly
Reduction branch-rot caused by lose, the development of biocontrol reagent is imperative.In recent years, grinding by biocontrol bacterial strain prevention and control disease
Study carefully relatively broad, Yang Hai is clear etc., which is found in walnut rhizosphere antagonistic bacterium, can preferably inhibit peach brown rot, and demonstrate short of money
Antibacterial antibacterial substance tool feature that bacteriostatic activity is strong, stability is good.Wang Yang etc. passes through tissue isolation and plate face-off training
The method of supporting filters out one plant of bacterial strain to Rot in walnut with stronger antagonistic effect, is finally accredited as bacillus subtilis
Bacillus subtilis。
The one kind of antagonistic bacterium as biological control, has a good application prospect to control of plant disease, it is domestic about
Correlative study was carried out in the biological control of walnut disease.The researchs such as Ge Kangkang are found to hickory nut dry rot pathogen
(Botryosphaeria dothidea) has the biocontrol microorganisms bacillus subtilis (Bacillus subtilis) of antagonistic effect.
Zhang Jingjing, Li Jiangui etc. filter out pseudomonas Pseudomonas sp., bacillus Bacillus sp. and streptomycete
Belong to Streptomyces sp. totally 7 plants of antagonism Rot in walnut bacterial strains.The Endophyte of walnut XHE7 of this heavy equal screening of Fu can promote
The root long and bud of seed are long.
Walnut branch-rot (Juglanconis juglandina) is in recent years on Xinjiang walnut branch, fruit and blade
Newfound one kind disease, there has been no the correlations for the biocontrol bacterial strain for capableing of antagonism Juglanconis juglandina to grind at present
Study carefully.
Walnut has become the leading industry of Xinjiang South Sinkiang rural economic development and increasing peasant income, therefore, withered for Ramulus Juglandis
Disease is research object, and there is an urgent need to develop the excellent bacterial strains of a collection of Biocontrol Effect, the biological control for walnut branch-rot.
Summary of the invention
The object of the present invention is to provide a kind of pair of walnut branch-rot germ (Juglanconis juglandina) have compared with
The bacillus subtilis (Bacillus subtilis) of good bacteriostasis and its application.
In order to achieve the object of the present invention, bacillus subtilis of the invention (Bacillus subtilis) is from Xinjiang
It is isolated in the diversiform-leaved poplar rot disease disease sample of autonomous region, Uygur acquisition, it is named as QB002.Through morphological feature, physiology
Biochemical character and 16S rDNA gene sequencing, specifying antagonistic strain QB002 is bacillus subtilis (Bacillus
subtilis).The bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms on July 12nd, 2019
Center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode
100101), classification naming is bacillus subtilis Bacillus subtilis, and deposit number is CGMCC No.18218.
The Microbiological Characteristics of bacillus subtilis (Bacillus subtilis) QB002 are as follows: bacterial strain QB002 is trained in NA
It supports after being cultivated for 24 hours on base, single colonie has fold in faint yellow, in irregular shape, intermediate, and opaque, quality is wet, and surface is not only
It is sliding.Purple is presented in Gram's staining, is gram-positive bacteria, and microexamination thallus is rod-short.
Physio-biochemical characteristics show that bacterial strain is gram-positive anaerobic bacterium；It can be 23 kinds to D- turanose, D- stachyose etc.
Carbon source is utilized；Catalase test is positive；Phenylalanine enzyme test, Tyrosinase Assay and the test of anti-nitrate reductase are in yin
Property；
The present invention provides the microbial inoculum containing the bacillus subtilis QB002.
The tunning of bacillus subtilis QB002 belongs to the scope of protection of the present invention.
The present invention provides produce containing bacillus subtilis (Bacillus subtilis) QB002 or its fermentation
The microbial inoculum of object.The microbial inoculum is the microbial inoculum of single bacterium, can also be the microbial inoculum of compound bacteria.
The present invention provides contain the bacillus subtilis (Bacillus subtilis) QB002 or its tunning
Drug.
The drug is to inhibit walnut branch-rot bacterium (Juglanconis juglandina), Valsa mali
(Cytospora salicicola, Cytospora mali), jujube fruit alternaria (Alternaria alternata) and Lee
The drug of brown rot germ (Monilinia fructicola).
The present invention provides the bacillus subtilis (Bacillus subtilis) QB002, its tunning or contain
There is the application of its microbial inoculum, drug in control plant disease.
Plant of the present invention includes but is not limited to walnut, Xinjiang poplar, lombardy poplar, diversiform-leaved poplar, willow, apple, birch, arrow-leaved oleaster
Or grape.
The present invention provides the bacillus subtilis (Bacillus subtilis) QB002, its tunning or contain
There are its microbial inoculum, drug preventing and treating walnut branch-rot bacterium (Juglanconis juglandina), Valsa mali
(Cytospora salicicola, Cytospora mali), jujube fruit alternaria (Alternaria alternata) and Lee
Application in brown rot germ (Monilinia fructicola).
Bacillus subtilis QB002 provided by the invention is able to suppress the growth of walnut branch-rot bacterium mycelia, causes mycelia abnormal
Shape, distortion generate multiple branches, and it is 88.6% to the inhibiting rate of walnut branch-rot that plate streak, which measures it, withered to Ramulus Juglandis
Germ (Juglanconis juglandina), canker of apple fruit (Cytospora salicicola, Cytospora mali),
Jujube fruit black spot (Alternaria alternata), Lee's brown rot (Monilinia fructicola) have certain inhibition to make
With with broad-spectrum antibacterial ability；Bacillus subtilis QB002 provided by the invention is latent with good biological and ecological methods to prevent plant disease, pests, and erosion to walnut branch-rot
Power can be used for preventing and treating walnut branch-rot, have the features such as pollution-free, noresidue, biological environmental production, in terms of the prevention and control of plant disease
With significant effect.
Detailed description of the invention
Fig. 1 is walnut branch-rot field hazard symptoms, and A figure shows aggrieved limb, and B figure shows aggrieved blade, C figure show by
Evil fruit.
The A figure of Fig. 2 is bacterial strain QB002 colonial morphology, and B figure is QB002 bacterium Gram's staining, is shown as purple.
Fig. 3 is the phylogenetic tree of bacillus subtilis (Bacillus subtilis) QB002.
Fig. 4 is 1 bacterial strain QB002 of embodiment to the fungistatic effect figure of different germs, and wherein A figure is the core inhibited by QB002
The colonial morphology of peach branch blight bacterium (Juglanconis juglandina)；B figure is the walnut branch-rot bacterium of normal growth
(Juglanconis juglandina) colonial morphology；C figure is the Valsa mali (Cytospora inhibited by QB002
Mali colonial morphology)；D figure is the colonial morphology of the Valsa mali (Cytospora mali) of normal growth；E figure be by
The colonial morphology for the Valsa mali (Cytospora salicicola) that QB002 inhibits；F figure is that the apple of normal growth is rotten
The colonial morphology of rotten germ (Cytospora salicicola)；G figure is the jujube fruit alternaria inhibited by QB002
The colonial morphology of (Alternaria alternata)；H is the jujube fruit alternaria (Alternaria of normal growth
Alternata colonial morphology)；I figure is the bacterium colony by the QB002 Lee's brown rot germ (Monilinia fructicola) inhibited
Form；J figure is the colonial morphology of Lee's brown rot germ (Monilinia fructicola) of normal growth.
Fig. 5 is microscopically observation bacillus subtilis (Bacillus subtilis) QB002 to pathogen
The influence of Juglanconis juglandina mycelia, left figure are the Juglanconis juglandina after being inhibited by QB002
Mycelia, right figure be normal pathogen Juglanconis juglandina mycelia.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means.Material as used in the following examples, examination
Agent etc. is commercially available unless otherwise specified.
Walnut branch-rot pathogen (Juglanconis juglandina) as used in the following examples is by Xinjiang agriculture
Industry University Forestry and College of Horticulture's Pathology Lab provide, and are isolated from the aggrieved tissue of Xinjiang Yili of China Gongliu County Chinese walnut, number
1429。
In embodiment, the culture of walnut branch-rot pathogen and plate face-off experiment use PDA culture medium: potato
200g, glucose 14g, agar 12g, water 1000mL, pH value are natural.
Bacteria Culture uses NA culture medium: beef extract 3.0g, peptone 10g, NaCl 5g, and agar 15g, distilled water are mended
To 1000mL, pH value 7.0 ± 0.1.
Antagonistic bacterium fermentation liquid use NB culture medium: beef extract 3.0g, peptone 10g, NaCl5g, distilled water mend to
1000mL, pH value 7.0 ± 0.1.
The separation and identification of 1 bacillus subtilis of embodiment (Bacillus subtilis) QB002
1, the separation of antagonistic bacterium
Disease sample is acquired in Xinjiang different regions, isolates bacterium bacterial strain, picking culture shape using tissue isolation
Different single colonie plate streakings on NA culture medium, 28 DEG C of constant temperature incubations for 24 hours after, to bacterium colony size, color, surface flatness
And edge shape etc. is observed and recorded, 4 DEG C of picking single colonie save backup.
2, the Identification of Species of antagonistic bacterium
The observation of 2.1 thalli morphologies and Biolog are analyzed and identified
Observe the culture form and Gram's staining of QB002 bacterial strain.It is single after bacterial strain QB002 is cultivated for 24 hours on NA culture medium
Bacterium colony has fold in faint yellow, in irregular shape, intermediate, and opaque, quality is wet, rough.Gram's staining is presented
Purple, is gram-positive bacteria, and thallus is rod-short (Fig. 2).
Physiological and biochemical property shows that bacterial strain QB002 is gram-positive anaerobic bacterium；It can be to D- turanose, D- stachyose etc.
23 kinds of carbon sources are utilized (table 1)；Catalase test is positive；Phenylalanine enzyme test, Tyrosinase Assay and anti-nitric acid are also
Original test is negative.
Purpose bacterial strain is identified according to the established procedure of the full-automatic assessing instrument of Biolog.It will be different using GP culture plate
The data of reaction are compareed with the data of known mode bacterial strain.Filtering out can in Biolog automatic identifying system database comparing result
Energy property (Probability) and similitude (Similarity) higher type, is finally accredited as Bacillus for bacterial strain QB002
subtilis。
1 bacterial strain QB002 of table is to 95 kinds of utilization of carbon source situations
Note: "+" is positive；"-" is negative；" b " is occupy between positive feminine gender.
Note:"+".Positive；"-" .Negative；"b".Intermediate.
The 16S rDNA Molecular Identification of 2.2 antagonistic strains
Bacterium total genomic dna is extracted by water-boiling method.
The amplification and sequencing of 16S rDNA: according to the conserved region of bacterial 16 S rDNA, PCR primer is designed.Using bacterium
Universal primer 63F (5 '-CAGGCCTAACACATGCAAGTC-3 ') and 1387R (5 '-GGGCGGWGTGTACAAGGC-3 ', wherein
W is to annex base A or T), primer is synthesized by Shanghai Sheng Gong bioengineering Co., Ltd.PCR reaction system (50 μ L): template DNA
45 μ L of μ L, 10 × Buffer of 2.5 μ L, primer each 2.5 μ L, dNTP, Taq polymerase 0.5 μ L, ddH2O supply 50 μ L.PCR is anti-
Answer condition: 94 DEG C of initial denaturation 3min；95 DEG C of denaturation 1min；55 DEG C of annealing 1min；72 DEG C of extension 1.5min, 30 circulations；72℃
Extend 10min.2 μ L PCR products are taken to detect PCR product with 1% agarose electrophoresis (120V/25min).Target fragment is by Shanghai
The sequencing of biotechnology Engineering Co., Ltd.Sequencing result is subjected to BLAST homologous sequence comparison on the website GenBank, is used
NJ (adjacent method) method phylogenetic tree construction in 6.0 software of MEGA, using method of bootstrapping (bootstrap) checking system tree, certainly
Opening up data set is 1000 times.Using Amphibacillus xylanus as outer group.
Obtained sequence is sequenced and carries out Blast comparison in NCBI, the results show that bacterial strain QB002 and B.subtilis relationship
Relationship is close, and homology 100% is shown on the phylogenetic tree constructed based on adjacent method: bacterial strain QB002 with
B.subtilis gathers in same branch and supporting rate with higher (99%) (Fig. 3), and the morphology of comprehensive bacterial strain and culture are special
Sign, utilization of carbon source situation and Molecular Phylogeny tree finally determine that bacterial strain QB002 is bacillus subtilis Bacillus
subtilis。
It is above-mentioned the experimental results showed that, bacterial strain QB002 be bacillus subtilis.The bacterial strain is in preservation on July 12 in 2019
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address: the Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101), classification naming is bacillus subtilis
(Bacillus subtilis), deposit number are CGMCC No.18218.
Growth inhibition effect of the 2 bacillus subtilis QB002 of embodiment to walnut branch-rot
Using plate opposite culture method measurement bacterial strain QB002 to the inhibiting effect of walnut branch-rot bacterium.Beating and taking diameter is 5mm
Purifying culture pathogen bacteria cake, be placed in PDA plate center.Bacterial strain QB002 is activated on NA culture medium, constant temperature training
It supports for 24 hours, picking single colonie is being crossed at pathogen 2cm, only to connect pathogen as control, 3 repetitions of every processing.28℃
Constant temperature incubation is observed when compareing bacterium colony and covering with plate, measures antibacterial bandwidth and pathogen growth diameter, and bacterium colony growth suppression is calculated
Rate processed, and filter out the optimal bacterium of antagonistic effect.
It is screened from 5 plants of antagonistic bacteriums through tablet face-off method and obtains 1 plant to walnut branch-rot bacterium (Juglanconis
Juglandina) the best bacterial strain QB002 (Fig. 4) of antagonistic effect, to the inhibiting rate of pathogen mycelia growth up to 88.6%.
The Primary Study of 3 bacillus subtilis QB002 antifungal mechanism of embodiment
Inhibition of the antagonistic strain QB002 active material to walnut branch-rot bacterium (Juglanconis juglandina) mycelia
Effect uses tablet face-off method, the pathogen bacteria cake that access diameter is 5mm in PDA plate center, in four away from plate center 1cm
Place sterile cover slips week, after mycelia growth, by bacterium QB002 scribing line at coverslip 1cm, 30 DEG C of culture 3d.With only
The mycelia of germ is connect as control, influence of the optical microphotograph microscopic observation antagonistic bacterium QB002 activated product to germ hypha form.
The result shows that antagonistic bacterium reaches 88.6% to the inhibiting rate of walnut branch-rot pathogen growth.Disease after being inhibited by antagonistic bacterium
Bacterium (Juglanconis juglandina) mycelia shows exception, is the most typically characterized in mycelia distortion, deformity, generates more
A branch's (left figure of Fig. 5).
The antimicrobial spectrum of 4 bacillus subtilis QB002 of embodiment measures
It is for trying germ, using tablet face-off method, not connect antagonism with canker of apple fruit, jujube fruit black spot, Lee's brown rot
Bacterium QB002 is control, and 3 repetitions of each processing, the constant temperature incubation at 28 DEG C, measurement is by short of money when compareing bacterium colony and covering with plate
The growth diameter of germ bacterium colony after antibacterial inhibition, calculates antibacterial percentage.The result shows that bacterial strain QB002 is not only to Ramulus Juglandis
Blight bacterium 1429-1 has inhibiting effect, and has certain inhibiting effect to canker of apple fruit, jujube fruit black spot, Lee's brown rot,
Antibacterial broad spectrum activity (Fig. 4) is shown, growth of pathogenic bacteria inhibiting rate is as shown in table 2.
Antibacterial result of the 2 bacterial strain QB002 of table to different germs
General explanation, specific embodiment and test have above been used, the present invention is described in detail, but
On the basis of the present invention, it can be modified or is improved, this is feasible to those skilled in the art.Therefore, In
Without departing from these modifications or improvements on the basis of spirit of that invention, fall within the scope of the claimed invention.
Sequence table
<110>Xinjiang Agricultural Univ
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Claims (9)
- Bacillus subtilis 1. (Bacillus subtilis) QB002, deposit number is CGMCC NO.18218.
- 2. the tunning of bacillus subtilis (Bacillus subtilis) QB002 described in claim 1.
- 3. the bacterium containing bacillus subtilis described in claim 1 (Bacillus subtilis) QB002 or its tunning Agent.
- 4. the medicine containing bacillus subtilis described in claim 1 (Bacillus subtilis) QB002 or its tunning Object.
- 5. drug as claimed in claim 4, which is characterized in that the drug is to walnut branch-rot bacterium (Juglanconis Juglandina), Valsa mali (Cytospora salicicola, Cytospora mali), jujube fruit alternaria (Alternaria alternata), Lee's brown rot germ (Monilinia fructicola) all have the drug of fungistatic effect.
- 6. bacillus subtilis (Bacillus subtilis) QB002, its tunning described in claim 1 contains it The application in control or controlling plant diseases of microbial inoculum, drug.
- 7. application as claimed in claim 6, which is characterized in that the plant is walnut, Xinjiang poplar, lombardy poplar, diversiform-leaved poplar, willow Tree, apple, birch, arrow-leaved oleaster or grape.
- 8. application as claimed in claim 6, which is characterized in that the plant disease is walnut branch-rot (Juglanconis Juglandina), canker of apple fruit (Cytospora salicicola, Cytospora mali), jujube fruit black spot (Alternaria alternata) and Lee's brown rot (Monilinia fructicola).
- 9. bacillus subtilis (Bacillus subtilis) QB002, its tunning described in claim 1 contains it Microbial inoculum, drug inhibit walnut branch-rot (Juglanconis juglandina) germ, canker of apple fruit (Cytospora Salicicola, Cytospora mali) germ, jujube fruit black spot (Alternaria alternata) germ, Lee's brown rot Application in (Monilinia fructicola) germ.
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Cited By (2)
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| CN112522154A (en) * | 2020-12-16 | 2021-03-19 | 黑龙江中医药大学 | Application of bacillus subtilis in biological prevention and control of phellodendron amurense rupr |
| CN113528377A (en) * | 2021-06-21 | 2021-10-22 | 山东碧蓝生物科技有限公司 | Bacillus subtilis with growth promoting and corrosion preventing effects and application thereof |
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| CN112522154A (en) * | 2020-12-16 | 2021-03-19 | 黑龙江中医药大学 | Application of bacillus subtilis in biological prevention and control of phellodendron amurense rupr |
| CN112522154B (en) * | 2020-12-16 | 2021-07-23 | 黑龙江中医药大学 | Application of bacillus subtilis in biological prevention and control of phellodendron amurense rupr |
| CN113528377A (en) * | 2021-06-21 | 2021-10-22 | 山东碧蓝生物科技有限公司 | Bacillus subtilis with growth promoting and corrosion preventing effects and application thereof |
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