CN105296396B - Endophytic Bacteria in Cotton HB3S-13 and its application in cotton verticillium wilt prevention - Google Patents
Endophytic Bacteria in Cotton HB3S-13 and its application in cotton verticillium wilt prevention Download PDFInfo
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Abstract
The present invention relates to Plant Pathologys, and in particular to Endophytic Bacteria in Cotton HB3S 13 and its application in cotton verticillium wilt prevention.The Endophytic Bacteria in Cotton HB3S 13 of the present invention, obtains, Classification And Nomenclature is from healthy cotton plant stem and root separation screening：（Agrobacterium sp.）HB3S 13, has been preserved in China typical culture collection center（CCTCC）, deposit number is：CCTCC NO:M 2015604, preservation date：On October 13rd, 2015.The HB3S 13 of the present invention can be cultivated on synthetic medium, be easy to get and cultivated, in favor of being factory produced and promoting and applying.The present invention Endophytic Bacteria in Cotton HB3S 13 to cotton verticillium wilt have favorable effect bacterium, control effect be 64.87~75.21%, and preventive effect stablize, in production apply more bacillus subtilis（Preventive effect is 46.0%~52.9%）It compares, control effect improves 50% or so.Biocontrol effect report still without the bacterium on cotton verticillium wilt in the prior art, becomes the biological prevention and control agent of business with larger potential development.
Description
Technical field
The present invention relates to Plant Pathologys, specifically, the present invention relates to Endophytic Bacteria in Cotton HB3S-13 and its in cotton
Application in verticillium wilt prevention.
Background technology
Cotton verticillium wilt (Verticillium wilt of cotton) is a kind of important disease on cotton, cotton yellow
Disease of withering is the silborne fungal diseases caused by verticillium dahliae (Verticillium dahliae K leb.), it can lead to cotton
Flower Severe Reduction even plant is dead.The conventional control method of cotton verticillium wilt, which has, cultivates disease-resistant variety, agricultural measures and chemistry
Prevention etc..Due to the variability of verticillium dahliae so that the cultivation and popularization of disease-resistant variety are restricted.Rice field-upland field rotation is proved to
It is a kind of more effective method, but it is to the more demanding of cotton field structure and water source.Chemical prevention and control method can be caused to environment
Pollution, and this method not only kills pathogen, also kills the beneficial microbe in soil, no matter the latter is to nature
Or the maintenance of agricultural soil system has important role.In consideration of it, people are dedicated to finding effective alternative.Biology
Prevention is a kind of method having application prospect, is prevented cotton verticillium wilt using biological control, can improve plant growth
With the safety for ensuring natural environment while yield, the strategic importance with sustainable development.
Have the characteristics that using the occurrence injury of beneficial microbe control plant disease economical, environmental-friendly.About with interior
Raw bacterium prevents the research of cotton verticillium wilt as biocontrol microorganisms, and domestic report is not also rarely seen, but realizes scale at present
Produce and can be used in the but and few of production practices, wherein a bacillus subtilis more successful example at last, life
The mechanism of action of object prevention is to generate a variety of antibacterial materials such as bacitracin to inhibit pathogen, to the anti-jig of verticillium wilt
There is certain effect, but there are many problems in production application, wherein most important problem is that control in field effect is relatively low,
The country registration bacillus subtilis dosage form to the control effect of cotton verticillium wilt 45% or so, and different year effect not
Stablize.It is biogenic pesticide in future therefore, it is necessary to excavate out the significantly more efficient microorganism resource of prevention to cotton verticillium wilt
Exploitation provides corresponding basis.
Endogenetic bacteria is that bacterium is unicellular as the advantage of biocontrol microorganisms, and growth and breeding is fast, convenient for large-scale fermentation
Production and preservation.The mechanism of bacterium controlling plant diseases mainly has antibiosis, the Competition of nutrition and site, hyperparasite to make
With and inducing plant resistance effect.
Invention content
Goal of the invention：The present inventor is directed to the deficiencies in the prior art, and the object of the present invention is to provide one
Kind Endophytic Bacteria in Cotton HB3S-13 carries out biological control with it to realize to cotton verticillium wilt.
Another object of the present invention is to provide Endophytic Bacteria in Cotton HB3S-13 described above in cotton verticillium wilt prevention
Application.
Technical solution：In order to realize that foregoing invention purpose of the present invention, the technical solution adopted by the present invention be：In a kind of cotton
Endophytic bacteria, Classification And Nomenclature are：(Agrobacterium sp.) HB3S-13, has been preserved in China typical culture collection center
(CCTCC), address：Wuhan, China Wuhan University, deposit number are：CCTCC NO:M 2015604, preservation date：2015
October 13.
The Endophytic Bacteria in Cotton HB3S-13 is obtained from healthy cotton plant stem and root separation screening to cotton yellow
Disease of withering has the endogenetic bacteria of preferable biocontrol effect and preventive effect effect stability, the endogenetic bacteria can be fixed well in cotton body
It grows, it is safe and non-toxic to people and animals, insect, plant, also there is following property：
1, morphological feature
Colonial morphologies of the bacterial strain HB3S-13 on NA culture mediums is faint yellow, and bacterium colony is circle, there is protrusion；To bacterial strain
HB3S-13 carries out Gram's staining, judges HB3S-13 for Gram-negative bacillus.
2,16S rRNA are analyzed
With the corresponding primer amplification of 16S rRNA ITS sequences, amplified production is purified and is sequenced, sequencing result with
Sequence on NCBI finds after being compared, the 16S rRNA ITS sequences of HB3S-13 endogenetic bacterias and rhizobium it is homologous
Property reaches 100%, therefore, HB3S-13 is attributed to rhizobium (Agrobacterium sp.).
In order to realize another object of the present invention, the present invention provides Endophytic Bacteria in Cotton HB3S-13 described above in cotton
Application in flower verticillium wilt prevention.
Beneficial effects of the present invention：
1, HB3S-13 can be cultivated on synthetic medium, be easy to get and cultivated, and the bacterial strain for biological control is necessary
It can be cultivated on synthetic medium, in favor of being factory produced and promoting and applying.
2, Endophytic Bacteria in Cotton HB3S-13 of the invention has cotton verticillium wilt the bacterium of favorable effect, prevention effect
Fruit is 64.87~75.21%, and preventive effect is stablized, to plant growth without toxic side effect, to people and animals, insect and crop safety, environment
Close friend, compared in production using more bacillus subtilis (preventive effect is 46.0%~52.9%), control effect improves
50% or so；In addition, this identified bacterium of Endophytic Bacteria in Cotton HB3S-13 of the present invention is Gram-negative bacillus, it is
Rhizobium (Agrobacterium sp.), still the biocontrol effect report without the bacterium on cotton verticillium wilt, have larger
Potential development becomes the biological prevention and control agent of business.
Description of the drawings
Fig. 1 is forms of the HB3S-13 on NA culture mediums；
Fig. 2 a, Fig. 2 b are respectively the aspect graph after Gram's staining HB3S-13；
Fig. 3 is comparison result figure of the 16S rRNA sequences of HB3S-13 on NCBI；
Fig. 4 is antagonism result figures of the HB3S-13 to verticillium dahliae；
The mixed liquor 1 that Fig. 5 is obtained after being mixed in equal volume with HB3S-13 bacterial solutions for verticillium dahliae spore liquid is to cotton seedling
The control effect figure of 30d after inoculation；
The mixed liquor 2 that Fig. 6 is obtained after being mixed in equal volume with HB3S-13 ferment products for verticillium dahliae spore liquid is to cotton
The control effect figure of 30d after seedling inoculation；
Fig. 7 is the cotton seedling growth control figure (positive control group picture) of 30d after being only inoculated with verticillium dahliae spore liquid；
Fig. 8 be HB3S-13 bacteriums figure with the control speed of growth of verticillium dahliae make-up culture compared with, wherein (a) for
The bacterium colony growing state of verticillium dahliae after HB3S-13 bacterium make-up cultures 14d, (b) be with it is big after blank ware make-up culture 14d
The bacterium colony growing state of beautiful Verticillium dahliae.
Endogenetic bacteria HB3S-13 was preserved in China typical culture collection center (CCTCC) on October 13rd, 2015,
Address：Wuhan, China Wuhan University, deposit number：CCTCC NO:M 2015604.
Specific implementation mode
The following is specific embodiments of the present invention, and technical scheme of the present invention is further described, but of the invention
Protection domain be not limited to these examples.It is every to be included in this hair without departing substantially from the change of present inventive concept or equivalent substitute
Within bright protection domain.
Culture medium prescription used in following embodiment is：
Czapek's medium formula：Potassium nitrate 2g, dipotassium hydrogen phosphate 1g, magnesium sulfate 0.5g, potassium chloride 0.5g, ferrous sulfate
0.01g, sucrose 30g, agar 15g, distilled water 1L, pH 7.3 ± 0.2；
PDA plate culture medium prescription：Potato 200g, sucrose 20g, agar 20g, distilled water 1L；
NA culture medium prescriptions：Beef extract 3g, peptone 10g, sodium chloride 5g, agar 20g, pH 7.3,
Distilled water 1L.
Embodiment 1
The separation method of cotton endophyte HB3S-13.
The separation process of cotton endophyte all carries out in super workbench, the stalk and main root mainly for cotton into
Row separation.The stalk of cotton and main root are cut into the plant fragments sample of 3cm long, then by the plant sample being cut into according to following
Program carries out surface sterilization:Using the alcohol disinfecting 1min of 70% (v/v), then with the hypochlorite disinfectant of 5% (v/v)
5min finally uses rinsed with sterile water 3~5 times again with the alcohol disinfecting 30s of 70% (v/v), each 3min.What disinfection was completed
Plant sample is placed on sterile dry filter paper, and the moisture on surface is blotted under aseptic condition.With the scalpel of disinfection by stalk and
Disinfectant is immersed at the sample both ends of root, and rest part is sliced with sterile surgical knife, and every about 2mm is thick.The sample sheet cut
On the PDA plate or NA culture mediums being positioned ready at once, 25 DEG C of cultures are placed in, the fungi of acquisition or bacterial clump pass through list
Spore or single bacterium silk are further purified.
2 endogenetic bacteria HB3S-13 of embodiment carries out Morphological Identification.
Colonial morphologies of the bacterial strain HB3S-13 on NA culture mediums is faint yellow, and bacterium colony is circle, there is protrusion；Thalline is rod-shaped,
See Fig. 1.Gram's staining is carried out to bacterial strain HB3S-13, it is judged for Gram-negative bacillus, form such as Fig. 2 a, schemes
Shown in 2b.
3 16S rRNA Molecular Identifications of embodiment.
Length with the corresponding primer amplification of 16S rRNA sequences, 16S rRNA is 1333bp, specific such as SEQ ID No.1
It is shown.
SEQ ID No.1
Sequence is surveyed after result is compared with the sequence on NCBI and is found, the 16S rRNA with Agrobacterium sp.
The homology of ITS sequence reaches 100%, and HB3S-13 is attributed to rhizobium (Agrobacterium sp.).Comprehensive HB3S-
13 morphological features, physiological characteristic and Molecular Identification by HB3S-13 as a result, be attributed to Agrobacterium sp..
Antagonisms (interior) of the 4 endogenetic bacteria HB3S-13 of embodiment to verticillium dahliae.
By fresh verticillium dahliae V225 pure culture biscuits involvng inoculations behind the center of PDA plate, 25 DEG C of culture 3d, draw respectively
50ul HB3S-13 bacterium solutions are placed in from four angles at colony edge 2cm, are observed two bacterium and be whether there is antagonism.Face-off training
It is foster that the results are shown in Figure 4, the results showed that, between two bacterium and antagonism, equal well-grown is not present.
Inhibiting effect of the 5 endogenetic bacteria HB3S-13 of embodiment to cotton verticillium wilt.
Verticillium dahliae V225 (defoliation bacterial strain) fungus block is inoculated into czapek's medium, 25 DEG C, 150rpm shake bacterium training
7~10d is supported, then with verticillium wilt pathogen spore liquid is obtained after 4 layers of filtered through gauze of sterilizing, the spore liquid concentration is 5 ╳ 106A spore
Son/ml, it is spare；
The bacterium solution of HB3S-13 is inoculated into NA culture mediums, 28 DEG C, 150rpm shake bacterium culture 3d after, use blood counting chamber
Adjustment bacterial concentration is 1 ╳ 108Then a/ml moves to bacterium solution in 50ml centrifuge tubes, 6000r/min centrifuges 10 points under room temperature
Clock, after centrifugation, supernatant is the metabolite of HB3S-13 bacterial strains, i.e. HB3S-13 ferment products；It will be obtained down after centrifugation
Layer precipitates sterile aqueous suspension, obtains HB3S-13 bacterial solutions；
Above-mentioned verticillium dahliae spore liquid and HB3S-13 bacterial solutions are obtained into mixed bacteria liquid 1 after mixing in equal volume, it will
Verticillium dahliae spore liquid carries out obtaining mixed bacteria liquid 2 after evenly mixing in equal volume with HB3S-13 ferment products, in addition also distinguishes
It regard verticillium dahliae spore liquid 3 and sterile water 4 as positive control and negative control group, follow-up cotton seedling is carried out at leaching root
Reason.
It is experimental cultivar with susceptible variety Ji cotton 11, after the cotton-seed ginning of susceptible variety Ji cotton 11, impregnates 1h, plant into cotton
In flower matrix, cotton seedling is removed from matrix when cotton seedling is grown to 1~2 true leaf, use to be seeded；The method of inoculation is i.e. by cotton seedling
Root immerse respectively in mixed bacteria liquid 1 described above, mixed bacteria liquid 2, verticillium dahliae spore liquid 3, sterile water 4, after 20min
It is planted in disposal plastic cup respectively with matrix again, 15 plants of cotton seedlings is managed per component other places, are divided in 5 plastic cups, each
3 young plants in cup.
15d institutes an inquiry disease index after inoculation, investigates disease index 25d to after being inoculated with every three days, calculates
Disease index, diseased plant rate and control effect.Experiment is repeated 3 times, and test result is as shown in Fig. 5, Fig. 6, Fig. 7, Disease investigation knot
Fruit is shown in Table one and table two, the results showed that, the disease index of positive control is significantly larger than HB3S-13 treated that disease refers to, sterile water
Negative control is not fallen ill.It is repeated the result shows that HB3S-13 bacterial solutions are to the average control effect of cotton verticillium wilt
71.15%, HB3S-13 ferment product are 60.67% to the average control effect of cotton verticillium wilt.
Table one：Greenhouse state of an illness exponential case application form
Table two：Experiment preventive effect result table is repeated several times
Inhibiting effect of the volatile materials of 6 endogenetic bacteria HB3S-13 of embodiment to verticillium dahliae.
Using make-up cultivation, two, the culture dish of a diameter of 90mm is taken, is poured into respectively in ware bottom (a), ware bottom (b)
The PDA culture medium of 20ml, then by the PDA culture medium in verticillium dahliae pure culture biscuits involvng inoculation to ware bottom (b), by endogenetic bacteria
HB3S-13 bacterium solutions 100ul is uniformly coated in the PDA culture medium in ware bottom (a), and two ware make-ups are set with ParafilmTM
It is cultivated in 25 DEG C of constant incubators, not connect bacterium as a contrast in ware bottom (a).Experiment is in triplicate.Observe verticillium dahliae
Upgrowth situation, 8d, which starts to calculate colony diameter using crossing method, calculates growth speed, surveyed every 1d it is primary, until
14d.Verticillium dahliae is grown with the numerical computations HB3S-13 endogenetic bacterias Volatile Metabolites measured by 14d
Inhibiting rate, the upgrowth situation of verticillium dahliae is shown in Fig. 8 (b) after 14 days.In addition, one group of control experiment of design, prepares above-mentioned identical
The culture dish of size carries out make-up culture to verticillium dahliae by above-mentioned same step, differs only in ware bottom (a) and be not coated with
Endogenetic bacteria HB3S-13 bacterium solutions.
Inhibiting rate=(control average diameter-processing average diameter)/control average diameter × 100%.It the results are shown in Table three.
The result shows that the volatile materials that HB3S-13 bacterium solutions generate can inhibit the growth of verticillium wilt pathogen, inhibiting rate to be
52%.
Table three：HB3S-13 and verticillium dahliae growing state comparison sheet in verticillium dahliae make-up culture group and control group
Influences of the 7 endogenetic bacteria HB3S-13 of embodiment to cotton seeds emergence rate.
It is experimental cultivar with susceptible variety Ji cotton 11, after the seed lint of susceptible variety Ji cotton 11, is immersed in HB3S-13
After impregnating 12h in bacterium solution, plant in cotton matrix；As a control group with the seed of the processed Ji cotton of clear water 11, it is calculated after 3d
Its emergence rate.The result shows that HB3S-13 bacterium solutions treated seeding ratio is 68.67%, treated that seed is (right for clear water
According to group) emergence rate be 75.5%, illustrate, influence of the HB3S-13 endogenetic bacterias to seeding ratio is small.
Claims (3)
1. Endophytic Bacteria in Cotton HB3S-13, Classification And Nomenclature are（Agrobacterium sp.）HB3S-13 has been preserved in China
Type Tissue Collection（CCTCC）, deposit number is：CCTCC NO:M 2015604, preservation date：2015 10
The moon 13.
2. Endophytic Bacteria in Cotton HB3S-13 described in claim 1 is used to control the application of cotton verticillium wilt.
3. Endophytic Bacteria in Cotton HB3S-13 described in claim 1 is used to inhibit the application of verticillium dahliae.
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| CN113151043B (en) * | 2021-02-04 | 2023-03-24 | 塔里木大学 | Rhizobium DG3-1 for resisting cotton verticillium wilt and application thereof |
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| CN102533603A (en) * | 2012-01-10 | 2012-07-04 | 南京农业大学 | Pseudomonas sp. 841P-3 for preventing cotton verticillium wilt and use thereof |
| CN102443559B (en) * | 2012-01-10 | 2013-04-10 | 南京农业大学 | Baclillus subtilis used for controlling cotton verticillium wilt and application thereof |
| CN103773708A (en) * | 2012-11-16 | 2014-05-07 | 江苏省农业科学院 | Disease prevention and growth promotion endophytic bacteria for plants |
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|---|---|---|---|---|
| CN102533603A (en) * | 2012-01-10 | 2012-07-04 | 南京农业大学 | Pseudomonas sp. 841P-3 for preventing cotton verticillium wilt and use thereof |
| CN102443559B (en) * | 2012-01-10 | 2013-04-10 | 南京农业大学 | Baclillus subtilis used for controlling cotton verticillium wilt and application thereof |
| CN103773708A (en) * | 2012-11-16 | 2014-05-07 | 江苏省农业科学院 | Disease prevention and growth promotion endophytic bacteria for plants |
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