CN105969707B - For preventing and treating the escherichia coli and its application of bacterial wilt - Google Patents
For preventing and treating the escherichia coli and its application of bacterial wilt Download PDFInfo
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- CN105969707B CN105969707B CN201610614212.8A CN201610614212A CN105969707B CN 105969707 B CN105969707 B CN 105969707B CN 201610614212 A CN201610614212 A CN 201610614212A CN 105969707 B CN105969707 B CN 105969707B
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Abstract
The invention discloses one plant for preventing and treating the escherichia coli and its application of bacterial wilt.Entitled escherichia coli (Escherichia coli) GZ-34 of the bacterial strain, deposit number are CCTCC NO:M 2016353, are preserved in the China typical culture collection center positioned at Wuhan, China Wuhan University on June 27th, 2016.Bacterial strain of the invention has significant antagonism to Ralstonia solanacearum, provides a kind of approach again for the prevention and treatment of bacterial wilt.
Description
Technical field
The present invention relates to agricultural biological technical fields, and in particular to one plant for prevent and treat bacterial wilt escherichia coli and
It is applied.
Background technique
Vegetative bacteria bacterial wilt is a kind of dimension as caused by green withered Raul Salmonella (Ralstonia solanacearum)
The crushing soil of tube bank passes bacterial disease, and the destructiveness soil that prevalent systems infect occurs in subtropical zone, the torrid zone and Temperate Region in China and passes
Disease is commonly called as " plant seasonal febrile diseases ".Green withered Raul Salmonella host is extensive, can infect more than 450 plants of 54 sections.Currently, bacterium
Property bacterial wilt prevention and treatment be universally acknowledged a great problem, it is distributed widely in all over the world, and provinces and cities occur tight in south China
Weight, especially in Zhejiang, not only Solanaceae, melon aggrieved, the mulberry bacterial wilt that large area occurs more cause sternly sericultural production in recent years
It loses again.
Ralstonia solanacearum has many microspecies, is divided into 5 biological strains according to the host range of Ralstonia solanacearum, 1 main harm of microspecies is more
Number plant of Solanaceae, including tomato, potato, eggplant and tobacco etc., also endanger other section plants；Microspecies 2 can infect banana, in the sea
The plants such as health (Heliconais) and plantain；3 main harm potato of microspecies, weak can also infect tobacco and tomato；Microspecies 4 infect
Ginger and weak infect the other plants such as potato.The bacterial wilt bacterial strain for separately having researcher that will be separated to from south China mulberry tree
It is named as microspecies 5.The ralstonia solanacearum for infecting potato is mainly microspecies l and microspecies 3.
Currently, the control method of bacterial wilt mainly has enhancement of field management, cultivates disease-resistant variety, using chemical agent and applies
With biocontrol agent etc., but it was verified that field management is difficult to effectively control bacterial wilt；Time-consuming for the breeding of resistant material；
And chemical prevention pollution environment, the destruction ecological balance, and the long-time service of chemical agent easily makes pathogen develop drug resistance.Cause
This, is increasingly valued by people using biocontrol bacterial strain and its activated product prevention and treatment ginger bacterial wilt.A large number of studies show that portion
Dividing bacterium, actinomyces is comparatively ideal bacterial wilt Antagonistic Fungi.
Currently, escherichia coli itself is as the research of bacterial wilt biocontrol bacterium agent, there is not been reported.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide one plant for preventing and treating bacterial wilt
Escherichia coli.
Another object of the present invention is to provide described for preventing and treating the application of the escherichia coli of bacterial wilt.
The purpose of the invention is achieved by the following technical solution: one plant for preventing and treating the escherichia coli of bacterial wilt, name
Referred to as escherichia coli (Escherichia coli) GZ-34, deposit number was CCTCC NO:M 2016353, in 2016
June 27 was preserved in the China typical culture collection center positioned at Wuhan, China Wuhan University.
The colonial morphology of the escherichia coli for being used to prevent and treat bacterial wilt are as follows: strain cell is rod-shaped, flat in LB agar
Bacterium colony is round when cultivating on plate, neat in edge and rule, surface wettability, smooth, opaque.
The application of the escherichia coli in prevention and treatment bacterial wilt for preventing and treating bacterial wilt.
The application of the escherichia coli in prevention and treatment bacterial wilt for preventing and treating bacterial wilt, is by escherichia coli
It is applied in the soil of culture crops or is sprayed at the surface of crops.
The crops are the crops for being easy to be infected by Ralstonia solanacearum；Preferably solanaceous crops.
The crops can be the crops infected by Ralstonia solanacearum, or the crops not infected by Ralstonia solanacearum.
A kind of bacterial wilt biological control microbial inoculum, containing above-mentioned for preventing and treating the escherichia coli of bacterial wilt.
The present invention has the following advantages and effects with respect to the prior art: the present invention provides one plant for preventing and treating bacterial wilt
Escherichia coli, provide a kind of approach again for the prevention and treatment of bacterial wilt.
Detailed description of the invention
Fig. 1 be in the present invention bacterial strain GZ-34 to the antagonistic effect photo figure of Ralstonia solanacearum.
Fig. 2 is bacterial wilt Antagonistic Fungi biological and ecological methods to prevent plant disease, pests, and erosion experiment effect figure in the present invention.
Fig. 3 is the photo figure of bacterial strain GZ-34 single colonie in the present invention.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1: bacterium is isolated and purified
1. culture medium
LB agar medium (LB solid medium): peptone 10g, yeast extract 5g, NaCl 10g, agar 15g are used
H2O is settled to 1000mL, and pH 7.0-7.2,121 DEG C sterilize 20 minutes.
2. experimental procedure
(1) sample acquire: select representative place (bacterial wilt fall ill field in healthy plant root soil, place
Guangdong Province), topsoil is scalped, is taken apart from the soil under earth's surface in the soil layer of 5~10 centimetres of depth, eachly with soil working tool is adopted
Point adopts 5 samples, is fitted into bag and performs record.
(2) preparation of soil dilution liquid: weighing 10g soil, is put into the triangular flask equipped with 90mL sterile water and sufficiently vibrates,
The soil dilution liquid of 1:10 concentration is made.After grogs precipitating, 1mL supernatant is drawn, moves into the test tube for filling 9mL aqua sterilisa
In, the Soil Slurry of 1:100 concentration is made, and so on, the Soil Slurry of 1:1000 and 1:10000 is made, is made 10-2、10-3、10-4、10-5With 10-6The soil liquid again is spare.
(3) bacterium separates: selection 10-3、10-4、10-5With 10-6Four each 0.1mL of concentration, by diluted Soil Slurry point
It does not pour into the culture dish of the agar medium containing LB, spreading rod is sterilized in the abundant calcination of alcolhol burner flame, is coated with after cooling
Plate.The culture dish being inoculated with is inverted, three repetitions are arranged in 30 DEG C of constant temperature incubations.
(4) result: 384 plants of isolated bacterium.
Embodiment 2: plate antagonistic effect of the isolated bacterium to Ralstonia solanacearum
1. culture medium
TTC solid medium: 1. peptone 10g, acid hydrolyzed casein (Casein Acid Hydrolysate) 1g, Portugal
Grape sugar 5g, agar 15g use H2O is settled to 1000mL, pH6.8-7.2；121 DEG C sterilize 20 minutes, obtain solution I；②TTC(2,
3,5- triphenyltetrazolium chloride) solution of mass percent 1% is made into distilled water, with 0.22 μm of membrane filtration degerming, obtain
Solution II；3. when solution I is cooled to about 45 DEG C, 0.5mL solution II is added in every 100mL solution I, can inverted plate.
TTC fluid nutrient medium: with TTC solid medium, agar and TTC is not added.
2. experimental procedure
2.1 plate antagonistic effect --- primary dcreening operations
2.1.1 actication of culture
Ralstonia solanacearum and bacterium carry out actication of culture: Ralstonia solanacearum GMI1000 (ATCC) and isolated 384 plants of embodiment 1
Bacterium is switched to TTC solid medium tablets and LB agar medium plate, cultivates 2 days respectively at 28 DEG C or trains in 30 DEG C
It supports 1 day, it is spare.
2.1.2 the preparation of Ralstonia solanacearum plate
Shaking flask culture is carried out in the Ralstonia solanacearum access TTC fluid nutrient medium of activation, 28 DEG C, 200rpm is cultivated 2 days, obtains blueness
Withered fermented liquid.Every other day with TTC solid medium inverted plate, 0.1mL (OD is added after waiting culture mediums to solidify in each plate600About
2.0) Ralstonia solanacearum fermentation liquid, uniformly, drying is spare for coating, obtains Ralstonia solanacearum plate.
2.1.3 plate antagonistic effect primary dcreening operation
The 384 plants of bacterium single colonies activated with the toothpick picking of sterilizing, point connect on Ralstonia solanacearum plate, each board joint 30
The different Antagonistic Fungi of strain, 3 repetitions, 28 DEG C of cultures, for 24 hours, 36h and 48h difference observation experiment be as a result, main detection inhibition zone
Whether there is or not.
2.2 plate antagonistic effect --- secondary screenings
2.2.1 actication of culture
Same 2.1.1, activation selection primary dcreening operation have the separation of bacterial of inhibition zone.
2.2.2 the preparation of Ralstonia solanacearum plate
Shaking flask culture is carried out in the Ralstonia solanacearum access TTC fluid nutrient medium of activation, 28 DEG C, 200rpm ferments 2 days, obtains blueness
Withered fermented liquid.Every other day with TTC solid medium inverted plate, 0.1mL (OD is added after waiting culture mediums to solidify in each plate600About
2.0) Ralstonia solanacearum fermentation liquid, uniformly, punch (Φ 5mm) punches after drying, and each plate makes a hole spare for coating.
2.2.3 separation of bacterial liquid fermentation
At second day of Ralstonia solanacearum liquid submerged culture, the separation of bacterial of activation, which is transferred in LB liquid medium, carried out shaking flask
Culture, 30 DEG C, 200rpm cultivates 24 hours (OD600About 2.0).
2.2.4 plate antagonistic effect secondary screening
10 μ L separation of bacterial fermentation liquids are drawn with liquid-transfering gun to be put into the hole of Ralstonia solanacearum plate, are dried up, 3 repetitions, 28 DEG C
Culture.For 24 hours, 36h and 48h difference observation experiment is as a result, the presence or absence of main detection inhibition zone and size.
3. result
53 plants of bacterium of antagonistic effect are obtained by Primary Screening Test, secondary screening, inhibition zone are punched by plate antagonistic effect
It is more obvious, illustrates that antagonism is better.Secondary screening obtains 6 plants of the preferable bacterium of antagonistic effect, and bacterial strain GZ-34 is exactly wherein one plant.
Inhibition zone is obvious as seen from Figure 1, illustrates that bacterial strain GZ-34 has apparent antagonism to Ralstonia solanacearum.
Embodiment 3: the biological control effect test of bacterial strain GZ-34
In order to examine the biocontrol effect of bacterial strain GZ-34, bacterial strain GZ-34 fermentation liquid is subjected to biological and ecological methods to prevent plant disease, pests, and erosion to bacterial wilt in potting
Effect test.
1. experimental procedure
1.1 buy eggplant seedling
Buy the eggplant seedling cultivated in greenhouse.
1.2 eggplant transplantation of seedlings
Substrate soil is filled in flowerpot (specification 170mm*160mm), is slightly compressed, then every basin moves into 1 plant of eggplant seedling, after watering
Two fingers are slightly compressed in root, are cultivated in greenhouse.It is cultivated 4 weeks or so in flowerpot.
It is prepared by the activation of 1.3 Ralstonia solanacearums and Antagonistic Fungi and fermentation liquid
Ralstonia solanacearum GMI1000 and Antagonistic Fungi GZ-34 are activated respectively, then choose single colonie access TTC fluid nutrient medium and LB
Fluid nutrient medium shaking flask culture, 200rpm, respectively at 28 DEG C and 30 DEG C cultures, two days (OD600All about 2.5).
1.4 Ralstonia solanacearums and Antagonistic Fungi inoculation
2 control groups, i.e. CK1 and CK2 is arranged in this test.CK1: being not added any bacterium, and 5mLTTC fluid nutrient medium is only added；
CK2: only add 5mL Ralstonia solanacearum fermentation liquid, antagonism fermented liquid is not added.Bacterial strain GZ-34 processing: according to Ralstonia solanacearum: Antagonistic Fungi volume
Than doing 3 processing, 5 basins of each processing, every plant of root is first poured 5mL Ralstonia solanacearum fermentation liquid, then poured again for 1:1,1:2.5 and 1:5
5mL, 12.5mL, 25mL antagonism fermented liquid.
1.5 record
Record disease plant number and morbidity severity every two days.
2. test result
As shown in Fig. 2, the control group CK1 for only connecing TTC fluid nutrient medium does not fall ill, the death rate 0, directly after artificial infection
The control group CK2 of Ralstonia solanacearum GMI1000 fermentation liquid all falls ill, the death rate 100%, inoculation Ralstonia solanacearum GMI1000 fermentation liquid and
Processing group 1:1,1:2.5,1:5 death rate of Antagonistic Fungi GZ-34 fermentation liquid is respectively 40%, 40%, 20%, it can be seen that, it is short of money
Antibacterial GZ-34 fermentation liquor treatment can make eggplant plant bacterial wilt be effectively controlled.
The identification of 4 bacterial strain GZ-34 of embodiment
It is detected through microbiological analysis inspection center, Guangdong Province, detection foundation is " common bacteria system identification handbook " (east
Elegant pearl edits Science Press) " Berger bacterial identification manual " the 9th edition and " Molecular Cloning:A Laboratory guide " second edition (Huang Pei
2002 Science Press of Tang Yi), qualification result are as follows: the 16S rDNA sequence and Escherichia coli (large intestine of strain GZ-34
Escherichia) homology up to 99.37%, morphological feature and physio-biochemical characteristics and Escherichia coli (large intestine angstrom
Uncommon Salmonella) it is most like.Specific testing result is as follows:
1. features such as the form of the preservation strain, culture, physiology, biochemistry
Escherichia coli GZ-34: belong to escherichia coli (Escherichia coli)；It is grown in LB culture medium good
Good, strain cell is rod-shaped, and when cultivating on LB agar plate, bacterium colony is round, neat in edge and rule, surface wettability, smooth, bacterium
It falls opaque (as shown in Figure 3).
2. bacterial strain GZ-34 physical and chemical experiment
Experimental result is as shown in table 1:
Table 1
Note: "+" indicates positive, and "-" indicates negative.
3.16S rRNA sequence alignment
Bacterial strain GZ-34 16S rRNA result of gene sequence determination is as follows:
TGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGGGCGG
TGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCATGGAGTCG
AGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCGCGAGGTCGCTTCTCTTTGTATG
CGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGACTTGACGTCATCCCCACCTTCCTCCAGTTTA
TCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCTGGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTA
ATCCCAACATTTCACAACACGAGCTGACGACAGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACATTCTCATC
TCTGAAAACTTCCGTGGATGTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCT
TGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGC
TCCGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCT
GTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCCACCGGTATTCCTCCAGA
TCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACTCAAGCTTGCCAGTATCAGATGCAGT
TCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACCGCCTGCGTGCGCTTTACGCCCAGTAATTCCGATT
AACGCTTGCACCCTCCGTATTACCGCGGCTGCTGGCACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATGA
GCAAAGGTATTAACTTTACTCCCTTCCTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGC
ATGGCTGCATCAGGCTTGCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAG
TTCCAGTGTGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCTA
ATCCCATCTGGGCACATCCGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATGCGGTATTAGC
TACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGTCCGCCACTCGTCAGCAAA
GAAGCAAGCTGCTTCCTGTTACCGTTCGACTGCATGGTAGCT
Gained 16S rRNA gene order is carried out to BLAST on the website NCBI and compares analysis, is obtained and escherichia coli
The similarity of bacterium 16S rRNA is up to 99.37%, and morphological feature and physio-biochemical characteristics and escherichia coli are most like, therefore
From the point of view of 16S rRNA gene sequencing and morphological feature, physio-biochemical characteristics result, which can be classified as large intestine angstrom
Uncommon Salmonella (Escherichia coli).
GZ-34 is named as escherichia coli (Escherichia coli) GZ-34, deposit number is CCTCC NO:M
2016353, the China typical culture collection center positioned at Wuhan, China Wuhan University is preserved on June 27th, 2016.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (4)
1. one plant for preventing and treating the escherichia coli of bacterial wilt, it is characterised in that:
Entitled escherichia coli (Escherichia coli) GZ-34, deposit number are CCTCC NO:M2016353, in
On June 27th, 2016 is preserved in the China typical culture collection center positioned at Wuhan, China Wuhan University.
2. the escherichia coli described in claim 1 for preventing and treating bacterial wilt is in the bacterial wilt of prevention and treatment solanaceous crops infection
Application.
3. the escherichia coli according to claim 2 for preventing and treating bacterial wilt is withered in the blueness of prevention and treatment solanaceous crops infection
Application in disease, it is characterised in that:
It is the surface that escherichia coli is applied to in the soil of culture crops or is sprayed at crops.
4. a kind of bacterial wilt biological control microbial inoculum, it is characterised in that: contain the large intestine for being used to prevent and treat bacterial wilt described in claim 1
Escherichia.
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| CN101880673A (en) * | 2010-07-15 | 2010-11-10 | 福建农林大学 | Method for cultivating tobacco bacterial wilt resistance line by utilizing root-specific promoter |
| CN104911129A (en) * | 2015-06-10 | 2015-09-16 | 湖南泰谷生物科技股份有限公司 | Bacillus amyloliquefaciens strain for prevention and treatment of tomato bacterial wilt and microbial organic fertilizer thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880673A (en) * | 2010-07-15 | 2010-11-10 | 福建农林大学 | Method for cultivating tobacco bacterial wilt resistance line by utilizing root-specific promoter |
| CN104911129A (en) * | 2015-06-10 | 2015-09-16 | 湖南泰谷生物科技股份有限公司 | Bacillus amyloliquefaciens strain for prevention and treatment of tomato bacterial wilt and microbial organic fertilizer thereof |
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