CN105969707A - Escherichia coli for preventing and controlling bacterial wilt and application thereof - Google Patents

Escherichia coli for preventing and controlling bacterial wilt and application thereof Download PDF

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CN105969707A
CN105969707A CN201610614212.8A CN201610614212A CN105969707A CN 105969707 A CN105969707 A CN 105969707A CN 201610614212 A CN201610614212 A CN 201610614212A CN 105969707 A CN105969707 A CN 105969707A
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preventing
bacterial wilt
colon bacillus
crops
treating
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CN105969707B (en
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邓音乐
宋施豪
杨春喜
崔朝宇
董廷艳
孙秀云
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South China Agricultural University
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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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates

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Abstract

The invention discloses Escherichia coli for preventing and controlling bacterial wilt and application thereof. The strain is named as Escherichia coli GZ-34, with the preservation number of CCTCC NO: M2016353, is preserved at the China Center for Type Culture Collection of Wuhan university in China on June 27, 2016. The Escherichia coli has the obvious antagonism effect on ralstonia solanacearum, and a way is also provided for preventing and controlling the bacterial wilt.

Description

For preventing and treating colon bacillus and the application thereof of bacterial wilt
Technical field
The present invention relates to agricultural biological technical field, be specifically related to a strain and wish for the large intestine angstrom preventing and treating bacterial wilt Salmonella and application thereof.
Background technology
Vegetative bacteria bacterial wilt is caused by blue or green withered Raul Salmonella (Ralstonia solanacearum) Kind of vascular bundle destructiveness soil passes bacterial disease, and in subtropical zone, the torrid zone and Temperate Region in China generation prevalent systems invade The crushing soil-borne disease of dye, is commonly called as " plant pestilence ".Blue or green withered Raul Salmonella host is extensive, can infect 54 More than 450 kind plants of individual section.At present, the preventing and treating of bacterial wilt is universally acknowledged a great problem, it It is distributed widely in all over the world, occurs in south China provinces and cities serious, especially in Zhejiang, the most not only eggplant Section, melon being injured, sericultural production is more caused heavy losses by the Mulberry bacterial wilt that large area occurs.
Ralstonia solanacearum has a lot of microspecies, is divided into 5 biological strains according to the host range of Ralstonia solanacearum, and microspecies 1 are main Endanger most plant of Solanaceae, including Fructus Lycopersici esculenti, Rhizoma Solani tuber osi, Fructus Solani melongenae and Nicotiana tabacum L. etc., also endanger other section plants； Microspecies 2 can infect the plants such as Fructus Musae, Heliconias (Heliconais) and plantain；Microspecies 3 main harm Rhizoma Solani tuber osi, Also Nicotiana tabacum L. and Fructus Lycopersici esculenti can weak be infected；Microspecies 4 infect Rhizoma Zingiberis Recens and weak infect the other plants such as Rhizoma Solani tuber osi.Separately grind Study carefully the named microspecies of bacterial wilt bacterial strain 5 that personnel will be separated on south China mulberry.Infect the green grass or young crops of Rhizoma Solani tuber osi Rot bacterium is mainly microspecies l and microspecies 3.
At present, the prevention and controls of bacterial wilt mainly has enhancement of field management, cultivates disease-resistant variety, use chemistry Medicament and use biocontrol agent etc., but it was verified that field management is difficult to effectively control bacterial wilt occurs；Anti- The selection-breeding of property material is the longest；And chemical prevention is polluted environment, is destroyed ecological balance, and the length of chemical agent Phase use easily makes pathogen develop immunity to drugs.Therefore, utilize biocontrol bacterial strain and activated product preventing and treating Rhizoma Zingiberis Recens thereof blue or green Rot is increasingly subject to people's attention.Numerous studies show, part antibacterial, actinomycetes are comparatively ideal green grass or young crops Rot Antagonistic Fungi.
At present, there is not been reported as the research of bacterial wilt biocontrol bacterium agent for colon bacillus self.
Summary of the invention
The primary and foremost purpose of the present invention is that the shortcoming overcoming prior art is with not enough, it is provided that a strain is used for preventing and treating green grass or young crops The colon bacillus of rot.
Another object of the present invention is to provide described for preventing and treating the application of the colon bacillus of bacterial wilt.
The purpose of the present invention is achieved through the following technical solutions: a strain is for preventing and treating the E of bacterial wilt Bacterium, entitled colon bacillus (Escherichia coli) GZ-34, deposit number is CCTCC NO:M 2016353, the Chinese Typical Representative culture being positioned at Wuhan, China Wuhan University it is preserved on June 27th, 2016 Preservation center.
The colonial morphology of the described colon bacillus for preventing and treating bacterial wilt is: strain cell is shaft-like, at LB When cultivating on agar plate, bacterium colony is circular, neat in edge and rule, surface wettability, smooth, opaque.
The application in preventing and treating bacterial wilt of the described colon bacillus for preventing and treating bacterial wilt.
The application in preventing and treating bacterial wilt of the described colon bacillus for preventing and treating bacterial wilt, is by large intestine angstrom Uncommon Salmonella puts in the soil cultivating crops or is sprayed at the surface of crops.
Described crops are the crops being prone to be infected by Ralstonia solanacearum；It is preferably solanaceous crops.
Described crops can be the crops infected by Ralstonia solanacearum, or the farming do not infected by Ralstonia solanacearum Thing.
A kind of bacterial wilt Biological control microbial inoculum, containing the above-mentioned colon bacillus for preventing and treating bacterial wilt.
The present invention has such advantages as relative to prior art and effect: the present invention provides a strain for preventing and treating The colon bacillus of bacterial wilt, the preventing and treating for bacterial wilt provides again a kind of approach.
Accompanying drawing explanation
Fig. 1 is the bacterial strain GZ-34 antagonistic effect photo figure to Ralstonia solanacearum in the present invention.
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 the mono-bacterium colony of bacterial strain GZ-34 in the present invention.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention It is not limited to this.
Embodiment 1: antibacterial carries out isolated and purified
1. culture medium
LB agar culture medium (LB solid medium): peptone 10g, yeast extract 5g, NaCl 10g, Agar 15g, uses H2O is settled to 1000mL, pH 7.0-7.2,121 DEG C of sterilizings 20 minutes.
2. experimental procedure
(1) sample collecting: select representative place (bacterial wilt morbidity field in healthy plant Soil-in-Root Earth, place Guangdong Province), scalp topsoil, take 5～10 centimetres of degree of depth under distance earth's surface with adopting soil working tool Soil in soil layer, each place is adopted 5 samples, is loaded in bag and perform record.
(2) preparation of soil dilution liquid: weigh 10g soil, puts into the triangular flask equipped with 90mL sterilized water In fully vibrate, make the soil dilution liquid of 1:10 concentration.After grogs precipitates, draw 1mL supernatant, Move in the test tube filling 9mL aquesterilisa, make the Soil Slurry of 1:100 concentration, the like, Make the Soil Slurry of 1:1000 and 1:10000, make 10-2、10-3、10-4、10-5With 10-6Times The soil liquid standby.
(3) bacteria distribution: select 10-3、10-4、10-5With 10-6Four each 0.1mL of concentration, by dilution Soil Slurry is poured in the culture dish containing LB agar culture medium respectively, and spreading rod is abundant at alcohol burner flame Calcination sterilizing, after cooling spread plate.The culture dish inoculated is inverted, 30 DEG C of constant temperature culture, arranges Three repetitions.
(4) result: isolated antibacterial 384 strain.
Embodiment 2: the antibacterial of the isolated flat board antagonistic effect to Ralstonia solanacearum
1. culture medium
TTC solid medium: 1. peptone 10g, acid hydrolyzed casein (Casein Acid Hydrolysate) 1g, glucose 5g, agar 15g, use H2O is settled to 1000mL, pH6.8-7.2；121 DEG C of sterilizings 20 Minute, obtain solution I；2. TTC (2,3,5-TTC) is made into mass percent with distilled water The solution of 1%, degerming with 0.22 μm membrane filtration, obtain solution II；3. when solution I is cooled to about 45 DEG C, Every 100mL solution I adds 0.5mL solution II, can be down flat plate.
TTC fluid medium: with TTC solid medium, is not added with agar and TTC.
2. experimental procedure
2.1 flat board antagonistic effect primary dcreening operations
2.1.1 actication of culture
Ralstonia solanacearum and antibacterial carry out actication of culture: Ralstonia solanacearum GMI1000 (ATCC) and embodiment 1 separate To 384 strain antibacterials be switched to TTC solid medium flat board and LB agar culture medium flat board, respectively Cultivate 2 days in 28 DEG C or cultivate 1 day in 30 DEG C, standby.
2.1.2 the preparation of Ralstonia solanacearum flat board
The Ralstonia solanacearum of activation accesses and carries out shake-flask culture in TTC fluid medium, and 28 DEG C, 200rpm cultivates 2 My god, obtain Ralstonia solanacearum fermentation liquid.It is down flat plate every other day with TTC solid medium, each etc. after culture medium solidifying Flat board adds 0.1mL (OD600About 2.0) Ralstonia solanacearum fermentation liquid, coating uniformly, dries up standby, obtains Ralstonia solanacearum flat board.
2.1.3 flat board antagonistic effect primary dcreening operation
The 384 strain antibacterial list bacterium colonies activated with the toothpick picking of sterilizing, point is connected on Ralstonia solanacearum flat board, each The Antagonistic Fungi that board joint 30 strain is different, 3 repetitions, 28 DEG C of cultivations, 24h, 36h and 48h observe reality respectively Test result, the presence or absence of main detection inhibition zone.
2.2 flat board antagonistic effects sieve again
2.2.1 actication of culture
Same 2.1.1, activation selects primary dcreening operation to have the separation of bacterial of inhibition zone.
2.2.2 the preparation of Ralstonia solanacearum flat board
The Ralstonia solanacearum of activation accesses and carries out shake-flask culture in TTC fluid medium, 28 DEG C, 200rpm fermentation 2 My god, obtain Ralstonia solanacearum fermentation liquid.It is down flat plate every other day with TTC solid medium, each etc. after culture medium solidifying Flat board adds 0.1mL (OD600About 2.0) Ralstonia solanacearum fermentation liquid, coating uniformly, dries up rear card punch (Φ 5mm) Punching, each flat board makes a hole standby.
2.2.3 separation of bacterial liquid fermentation
At second day of Ralstonia solanacearum liquid submerged culture, the separation of bacterial of activation proceeded to enter in LB fluid medium Row shake-flask culture, 30 DEG C, 200rpm cultivates 24 hours (OD600About 2.0).
2.2.4 flat board antagonistic effect sieves again
Draw in the hole that Ralstonia solanacearum flat board put into by 10 μ L separation of bacterial fermentation liquids with liquid-transfering gun, dry up, 3 weights Multiple, 28 DEG C of cultivations.24h, 36h and 48h respectively observation experiment result, the presence or absence of main detection inhibition zone with Size.
3. result
Antibacterial 53 strain having antagonistic effect is obtained by Primary Screening Test, by the multiple sieve of flat board antagonistic effect punching, Inhibition zone is the most obvious, illustrates that antagonism is the best.Multiple sieve obtains antagonistic effect preferable antibacterial 6 strain, bacterial strain GZ-34 is exactly a wherein strain.
Inhibition zone is obvious as seen from Figure 1, illustrates that bacterial strain GZ-34 has obvious antagonism and makees Ralstonia solanacearum With.
Embodiment 3: the Biological control effect test of bacterial strain GZ-34
In order to check the biocontrol effect of bacterial strain GZ-34, by bacterial strain GZ-34 fermentation liquid on potted plant to bacterial wilt Carry out biocontrol effect test.
1. experimental procedure
1.1 buy Fructus Solani melongenae Seedling
Buy the Fructus Solani melongenae Seedling cultivated in booth.
1.2 Fructus Solani melongenae transplantation of seedlings
Filling substrate soil at flowerpot (specification 170mm*160mm), slightly compress, then every basin moves into 1 strain Fructus Solani melongenae Seedling, after watering, two refer to slightly compress at root, cultivate in booth.Flowerpot is cultivated about 4 weeks.
The activation of 1.3 Ralstonia solanacearums and Antagonistic Fungi is prepared with fermentation liquid
Ralstonia solanacearum GMI1000 and Antagonistic Fungi GZ-34 activates respectively, then chooses single bacterium colony and accesses TTC liquid Culture medium and LB fluid medium shake-flask culture, 200rpm, cultivates two days (OD respectively at 28 DEG C and 30 DEG C600 The most about 2.5).
1.4 Ralstonia solanacearums and Antagonistic Fungi inoculation
This test arranges 2 matched groups, i.e. CK1 and CK2.CK1: be not added with any bacterium, only add 5mL TTC fluid medium；CK2: only add 5mL Ralstonia solanacearum fermentation liquid, be not added with Antagonistic Fungi fermentation liquid.Bacterial strain GZ-34 Process: according to Ralstonia solanacearum: Antagonistic Fungi volume ratio is 1:1,1:2.5 and 1:5, do 3 process, each process 5 basins, every strain root first waters 5mL Ralstonia solanacearum fermentation liquid, waters 5mL, 12.5mL, 25mL antagonism the most again Fermented liquid.
1.5 record
Record disease plant number and the morbidity order of severity every two days.
2. result of the test
As in figure 2 it is shown, after artificial vaccination, the matched group CK1 only connecing TTC fluid medium does not falls ill, extremely Dying rate 0, the matched group CK2 of direct Ralstonia solanacearum GMI1000 fermentation liquid all falls ill, mortality rate 100%, Inoculation Ralstonia solanacearum GMI1000 fermentation liquid and process group 1:1 of Antagonistic Fungi GZ-34 fermentation liquid, 1:2.5,1:5 are dead The rate of dying is respectively 40%, 40%, 20%, it can be seen that, Antagonistic Fungi GZ-34 fermentation liquor treatment can make Fructus Solani melongenae plant Strain bacterial wilt is effectively controlled.
The qualification of embodiment 4 bacterial strain GZ-34
Detecting through microbiological analysis inspection center of Guangdong Province, its detection foundation is " common bacteria system identification hands Volume " (east elegant pearl chief editor Science Press) " the outstanding Bacteria Identification handbook of uncle " the 9th edition and " molecular cloning Experiment guide " second edition (Huang Peitangyi 2002 Science Press), qualification result is: strain GZ-34's 16S rDNA sequence reaches 99.37% with the homology of Escherichia coli (colon bacillus), and its form is special Levy and physio-biochemical characteristics are most like with Escherichia coli (colon bacillus).Concrete testing result is such as Under:
1. the feature such as the form of this preservation strain, cultivation, physiology, biochemistry
Colon bacillus GZ-34: belong to colon bacillus (Escherichia coli)；In LB culture medium Well-grown, strain cell is shaft-like, and when cultivating on LB agar plate, bacterium colony is circular, neat in edge and rule, Surface wettability, smooth, bacterium colony 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: "+" representing the positive, "-" represents negative.
3.16S rRNA sequence alignment
Bacterial strain GZ-34 16S rRNA gene sequencing result is as follows:
TGGTAGCGCCCTCCCGAAGGTTAAGCTACCTACTTCTTTTGCAACCCACTCCCATGGTGTGACGG GCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGA CTTCATGGAGTCGAGTTGCAGACTCCAATCCGGACTACGACGCACTTTATGAGGTCCGCTTGCTCTCG CGAGGTCGCTTCTCTTTGTATGCGCCATTGTAGCACGTGTGTAGCCCTGGTCGTAAGGGCCATGATGA CTTGACGTCATCCCCACCTTCCTCCAGTTTATCACTGGCAGTCTCCTTTGAGTTCCCGGCCGGACCGCT GGCAACAAAGGATAAGGGTTGCGCTCGTTGCGGGACTTAATCCCAACATTTCACAACACGAGCTGAC GACAGCCATGCAGCACCTGTCTCACGGTTCCCGAAGGCACATTCTCATCTCTGAAAACTTCCGTGGAT GTCAAGACCAGGTAAGGTTCTTCGCGTTGCATCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCC CCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCCCAGGCGGTCGACTTAACGCGTTAGCTC CGGAAGCCACGCCTCAAGGGCACAACCTCCAAGTCGACATCGTTTACGGCGTGGACTACCAGGGTAT CTAATCCTGTTTGCTCCCCACGCTTTCGCACCTGAGCGTCAGTCTTCGTCCAGGGGGCCGCCTTCGCC ACCGGTATTCCTCCAGATCTCTACGCATTTCACCGCTACACCTGGAATTCTACCCCCCTCTACGAGACT CAAGCTTGCCAGTATCAGATGCAGTTCCCAGGTTGAGCCCGGGGATTTCACATCTGACTTAACAAACC GCCTGCGTGCGCTTTACGCCCAGTAATTCCGATTAACGCTTGCACCCTCCGTATTACCGCGGCTGCTGG CACGGAGTTAGCCGGTGCTTCTTCTGCGGGTAACGTCAATGAGCAAAGGTATTAACTTTACTCCCTTC CTCCCCGCTGAAAGTACTTTACAACCCGAAGGCCTTCTTCATACACGCGGCATGGCTGCATCAGGCTT GCGCCCATTGTGCAATATTCCCCACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTG TGGCTGGTCATCCTCTCAGACCAGCTAGGGATCGTCGCCTAGGTGAGCCGTTACCCCACCTACTAGCT AATCCCATCTGGGCACATCCGATGGCAAGAGGCCCGAAGGTCCCCCTCTTTGGTCTTGCGACGTTATG CGGTATTAGCTACCGTTTCCAGTAGTTATCCCCCTCCATCAGGCAGTTTCCCAGACATTACTCACCCGT CCGCCACTCGTCAGCAAAGAAGCAAGCTGCTTCCTGTTACCGTTCGACTGCATGGTAGCT
Gained 16S rRNA gene order is carried out BLAST comparison analysis on NCBI website, obtain with The similarity of colon bacillus 16S rRNA reaches 99.37%, and its morphological characteristic and physio-biochemical characteristics are with big Intestinal Escherichia is most like, therefore according to 16S rRNA gene sequencing and morphological characteristic, Physiology and biochemistry spy From the point of view of property result, this bacterial strain can be classified as colon bacillus (Escherichia coli).
By named for GZ-34 colon bacillus (Escherichia coli) GZ-34, deposit number is CCTCC NO:M 2016353, was preserved in the Chinese Typical Representative being positioned at Wuhan, China Wuhan University on June 27th, 2016 Culture collection center.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention are not by above-mentioned reality Execute the restriction of example, the change made under other any spirit without departing from the present invention and principle, modification, Substitute, combine, simplify, all should be the substitute mode of equivalence, within being included in protection scope of the present invention.

Claims (6)

1. a strain is for preventing and treating the colon bacillus of bacterial wilt, it is characterised in that: entitled colon bacillus (Escherichia coli) GZ-34, deposit number was CCTCC NO:M 2016353, in 2016 6 The moon is preserved in the China typical culture collection center being positioned at Wuhan, China Wuhan University on 27th.
2. the answering in preventing and treating bacterial wilt of the colon bacillus for preventing and treating bacterial wilt described in claim 1 With.
Colon bacillus for preventing and treating bacterial wilt the most according to claim 2 is in preventing and treating bacterial wilt Application, it is characterised in that: be colon bacillus is put on cultivate crops soil in or spray Surface crops.
Colon bacillus for preventing and treating bacterial wilt the most according to claim 3 is in preventing and treating bacterial wilt Application, it is characterised in that: described crops are the crops being prone to be infected by Ralstonia solanacearum.
Colon bacillus for preventing and treating bacterial wilt the most according to claim 4 is in preventing and treating bacterial wilt Application, it is characterised in that: described crops are solanaceous crops.
6. a bacterial wilt Biological control microbial inoculum, it is characterised in that: containing being used for described in claim 1 preventing and treating green grass or young crops The colon bacillus of rot.
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CN107384970A (en) * 2017-08-21 2017-11-24 华南农业大学 A kind of ETEC metabolism crude extract and preparation method and application
CN107467024A (en) * 2017-08-21 2017-12-15 华南农业大学 Applications of Cyclic dipeptides cyclo (S) pro (S) phe in preventing and treating bacterial wilt and/or rice blast

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107384970A (en) * 2017-08-21 2017-11-24 华南农业大学 A kind of ETEC metabolism crude extract and preparation method and application
CN107467024A (en) * 2017-08-21 2017-12-15 华南农业大学 Applications of Cyclic dipeptides cyclo (S) pro (S) phe in preventing and treating bacterial wilt and/or rice blast
CN107384970B (en) * 2017-08-21 2020-11-13 华南农业大学 Escherichia coli metabolism crude extract and preparation method and application thereof

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