CN113652377B - Bacterial strain for antagonizing pathogenic bacteria of tobacco target spot and application thereof - Google Patents
Bacterial strain for antagonizing pathogenic bacteria of tobacco target spot and application thereof Download PDFInfo
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- CN113652377B CN113652377B CN202111094181.5A CN202111094181A CN113652377B CN 113652377 B CN113652377 B CN 113652377B CN 202111094181 A CN202111094181 A CN 202111094181A CN 113652377 B CN113652377 B CN 113652377B
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Abstract
The invention discloses a strain for antagonizing pathogenic bacteria of tobacco target spot, which is pseudomonas aeruginosa GT-6, is preserved in China center for type culture collection, has the preservation date of 2021 year, 7 months and 30 days, and has the preservation number of CCTCC NO: m2021953, the preservation address is China, wuhan university, the taxonomy is named as Pseudomonas aeruginosa GT-6; the strain which antagonizes the pathogenic bacteria of the tobacco target spot is applied to the prevention and treatment of the tobacco target spot, so that the pathogenic bacteria can be prevented from generating drug resistance, and the prevention and treatment effect is improved.
Description
Technical Field
The invention relates to the technical field of strain screening, in particular to a strain for antagonizing pathogenic bacteria of tobacco target spot and application thereof.
Background
Tobacco target leaf spot is a fungal disease caused by dermataceae citrullina. The target spot of tobacco can occur from the seedling stage to the mature stage of tobacco leaves, which not only infects leaves, but also harms stems. The infected leaves are initially round water stain-shaped spots, if the temperature is high, the humidity is high, and the wetting time of the leaves is long, the scab rapidly expands to form irregular spots with the diameter of 2-20cm, concentric rings are formed to form chlorotic halo and withered spots, necrotic parts of the scab are fragile to form through holes, and the scab is shaped like a hole left on a target after bullet shooting, so the target spot is called. When the humidity is high, mycelium, sexual generation sporoderm and basidiospore of the fungus are often generated at the edge of lesion spots on the lower surface of the leaf.
At present, the prevention and treatment measures of the tobacco target spot disease are mainly chemical pesticide prevention and treatment, but the prevention and treatment by using a chemical agent have pesticide residues, seriously pollute the environment and are harmful to people and livestock, and the plant can generate certain drug resistance by using the same chemical pesticide for a long time, so the prevention and treatment effect is greatly reduced. The biological control can not pollute the environment, can not harm the health of people and livestock, and can effectively control the spread of the disease.
Therefore, how to provide a strain capable of antagonizing pathogenic bacteria of tobacco target spot is a technical problem to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of the above, the invention provides a strain for antagonizing pathogenic bacteria of tobacco target spot disease, and the strain is applied to control of the tobacco target spot disease.
In order to achieve the purpose, the invention adopts the following technical scheme:
the strain for antagonizing pathogenic bacteria of the tobacco target spot is pseudomonas aeruginosa GT-6, is preserved in China Center for Type Culture Collection (CCTCC) with the preservation date of 2021 year, 7 months and 30 days and the preservation number of CCTCC NO: m2021953, the preservation address is China, wuhan university, the taxonomy is named as Pseudomonas aeruginosa GT-6.
The application of the strain for antagonizing the pathogenic bacteria of the tobacco target spot disease in preventing and treating the tobacco target spot disease.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.
FIG. 1 is a diagram showing the results of the culture of GT-6 strain in accordance with the present invention in opposition to Rhizoctonia solani;
FIG. 2 is a diagram showing the bacteriostatic effect of the GT-6 strain on Rhizoctonia solani hyphae provided by the present invention;
FIG. 3 is a colony morphology of the GT-6 strain provided by the present invention;
FIG. 4 is a gel electrophoresis diagram of the 16S rDNA PCR product of the GT-6 strain provided by the present invention;
FIG. 5 is a diagram showing the results of homology alignment of the GT-6 strain provided in the present invention;
FIG. 6 is a phylogenetic tree result diagram of the GT-6 strain provided by the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Materials, reagents and instruments used in the examples:
tobacco rhizosphere soil: soil at the root of tobacco seedlings collected from 5 tobacco fields with serious tobacco target spots in Zhang Jiajiu city, yongshun county, longshan county, shimen county and Cili county in Hunan province.
Culture medium and medicament
LB medium: weighing 10g of peptone, 5g of yeast powder and 10g of NaCl by using an electronic balance, adding 500ml of deionized water, stirring by using a glass rod to completely dissolve the medicines, adding the deionized water to a constant volume of 1L, subpackaging 1L of culture medium in 500ml conical flasks, adding 200ml of culture medium in each flask, adding 1.5g of agar into each conical flask, carrying out moist heat sterilization at 121 ℃ for 25min by using a high-pressure steam sterilization pot, preparing an LB solid culture medium, and cooling for later use;
LB liquid medium: the preparation method is the same as above, but no agar is added, the culture medium is subpackaged into 50ml conical flasks, each flask is subpackaged with 20ml, and the culture medium is sterilized by moist heat at 121 ℃ for 25min in a high-pressure steam sterilization pot. Is configured into LB liquid culture medium;
medicament: peptone, yeast powder, nacl, agar powder, CTAB, absolute ethanol, phenol-chloroform-isoamyl alcohol (25;
laboratory apparatus
2L measuring cylinder, glass rod, 2ml centrifuge tube, 15ml centrifuge tube, pipette, coating rod, disposable culture dish, high pressure steam sterilization pot, super clean bench, shaking table, etc.
Example 1 Strain screening
1) Sampling
Digging healthy tobacco seedlings from tobacco fields with serious tobacco target spot diseases in the tobacco fields in various tobacco areas in Hunan, filling the tobacco seedlings into sterilized paper bags for storage, taking the tobacco seedlings back to a laboratory, shaking off soil at the root of the tobacco, and sieving coarse soil to leave fine soil.
2) Preparation of soil suspensions
Weighing soil sample 0.1g into 15ml centrifuge tube, adding sterile water 9.9ml to obtain 10% -2 g/ml of soil suspension, using a 1ml pipette to obtain a concentration of 10 -2 g/ml of the soil suspension in 9ml of sterile water to 10ml of 10 - 3 g/ml of soil suspension, according to which the soil suspension is further diluted to a concentration of 10 -6 g/ml、10 -7 g/ml and 10 -8 g/ml soil suspensionAnd (3) floating liquid, taking 200ul of each suspension with the three concentrations, adding the suspensions to a PDA culture medium and an LB culture medium respectively for coating and bacteria division, coating three culture dishes on each suspension with the three concentrations, putting the coated PDA culture dish in an incubator at 28 ℃, putting the coated LB culture dish in an incubator at 37 ℃, and carrying out inverted culture on the two culture dishes with different media. Observing the growth of the strains under different dilution concentrations after a period of time, selecting proper single colonies from the screened bacteria for streaking and purifying, selecting hypha from the screened fungi or beating a fungus cake at the edge of the single colony for purifying, numbering the separated strains, and storing at 4 ℃ for next step of antagonistic experiment identification. The co-isolation yielded 33 strains, of which 23 strains of bacteria, 7 strains of fungi and 3 strains of actinomycetes.
3) Screening of biocontrol bacteria
Using tobacco target leaf spot pathogen (R.solani) as a target, and carrying out opposite culture on a strain separated from tobacco rhizosphere soil and Rsolani, wherein the specific steps are as follows: inoculating the activated bacterial cake of the tobacco target leaf spot germs on a PDA culture dish, wherein the position is about 1cm close to the edge of the culture dish, and then inoculating the screened bacteria and fungi on the opposite side of the Rsolani, and the distance between the screened bacteria and fungi and the edge of the PDA culture dish is also about 1 cm. The inoculation of the screened bacteria and actinomycetes is carried out by utilizing a single colony generated by streaking, the single colony is inoculated on the opposite side of the Rsolani by utilizing a sterilized toothpick, and the screened fungi are inoculated on the edge of the fungal colony in a way of beating a fungus cake and then are inoculated on the opposite side of the Rsolani. And (3) carrying out confronting culture on all the screened microorganisms and R.solani according to the method, inoculating three times of each strain, inoculating a sterile PDA strain cake and inoculating sterile water to serve as a control group, putting the inoculated PDA culture dish into an incubator at 28 ℃ for culture, and observing after culturing for a period of time. Screening out biocontrol bacteria with obvious inhibition effect on the growth of hyphae of the tobacco target leaf spot pathogen, wherein the numbers are GT-6 respectively, and the results are shown in figure 1;
4) Determination of biocontrol bacterium inhibition rate
Selecting the separated substance with the bacteriostatic effect obtained by screening, inoculating the Rsolani bacteria in the center of a PDA culture dish, inoculating microorganisms with the bacteriostatic effect around the Rsolani bacteria cake, beating the bacteria cake of the fungus separated substance for inoculation, and inoculating four points for three times, wherein a control group is set to be inoculated with sterile PDA bacteria cake and sterile water and is placed in an incubator at 28 ℃ for culture, when the Rsolani hyphae of the control group overgrow the antibacterial dish, the diameter of the Rsolani after inoculation antagonism is measured, and the bacteriostatic rate of the antagonism bacteria is calculated according to the diameter (cm) of a processing group and the diameter (cm) of the control group. The bacteriostatic rate (100%) = (control colony diameter-treated colony diameter)/control colony diameter × 100%.
After the rhizoctonia solani of the control group grows in a culture dish with the diameter of 90mm, measuring the colony diameter of the rhizoctonia solani after inoculation of biocontrol bacteria by using a vernier caliper, setting three times of repetition, calculating the average diameter (mm) of the colony after biocontrol bacteria treatment, and calculating the bacteriostasis rate of the strain GT-6 to be 63.98 percent as shown in a figure 2 and a table 1;
TABLE 1
5) Identification of biocontrol strain
Morphological observation
The GT-6 bacterial colony is round, has smooth edge, is gray green in color and has metallic luster, and is shown in figure 3;
(1) Extraction of biocontrol bacteria DNA
And (3) performing molecular identification on the screened strain with the antagonistic effect, and extracting DNA of the strain by adopting an improved CTAB method.
Inoculating the purified fungi into a PDA culture dish filled with cellophane, inoculating each strain on the cellophane, culturing at 28 ℃ until hyphae grow over the culture dish, and hanging the hyphae in a 2ml centrifuge tube for later use;
1) Adding 500 μ l CTAB into 2ml centrifuge tube containing fungal hyphae, water bathing at 60 deg.C for 45min, and shaking every 15 min;
2) After completion of the water bath, 500. Mu.l of phenol-chloroform-isoamyl alcohol (25;
3) Chloroform-isopropanol (24
4) After the centrifugation is finished, taking the supernatant into a new centrifuge tube, adding chloroform-isoamylol (24;
5) Adding the supernatant into a new 2ml centrifugal tube, adding anhydrous ethanol with twice volume, and standing in a refrigerator at-20 deg.C for 30min;
6) Centrifuging at 12000rpm for 10min, pouring out liquid, adding 1ml of 75% absolute ethyl alcohol, centrifuging at 8000rpm for 5min, discarding supernatant, reversing the centrifugal tube on absorbent paper to volatilize residual liquid in the tube, adding 50 μ l of ultrapure water to dissolve DNA, and preserving at-20 deg.C for later use.
(2) PCR amplification of DNA
PCR amplification System and amplification procedure are as described in chapter II
The DNA extracted by CTAB method was used as a template, and 16S rDNA universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', shown in SEQ ID NO. 1) and 1492R (5'-CTACGGCTACCTTGTTACGA-3', shown in SEQ ID NO. 2) were used as primers (ordered from Shanghai Biotechnology Ltd.).
The PCR amplification system is shown in Table 2;
the procedure is as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 59 ℃ for 30s, extension at 72 ℃ for 1min, re-extension at 72 ℃ for 10min,30 cycles, and storing at 4 ℃.
TABLE 2 1696 rDNA Universal amplification System
(3) Sequencing of DNA samples
After the PCR products which are remained after the successful amplification sample application are written with the labels, the PCR products are sent to be sequenced, a segment of sequence obtained by sequencing is copied on NCBI to be subjected to BLAST comparison, and the comparison result is shown in figure 5; the phylogenetic tree was constructed using the NJ method using NCBI to download bacterial sequences from Pseudomonas and Bacillus velezensis as an outbreak, see FIG. 6.
The size of the strain GT-6 is 1421bp, the homology with the sequence of the Pseudomonas fluorescens with the accession number of MN736634.1 is up to 99 percent, and the biocontrol bacterium separated from the rhizosphere soil of the tobacco can be preliminarily determined to be Pseudomonas aeruginosa (Pseudomonas aeruginosa) according to phylogenetic tree analysis.
6) Evaluation of field control of strain GT-6 on tobacco target spot
2 mu of tobacco field with serious tobacco target spot disease is selected in Huayuan county of Hunan west, and 4 treatments are set in the experiment: 80% mancozeb wettable powder; 10% validamycin aqua, GT-6 fermentation liquor; media (control). Each process set 4 repetitions for 16 cells, each cell area about 50m2. The dosage of each treatment is as follows: 80% mancozeb wettable powder is diluted 1000 times and applied; the GT-6 fermentation liquor is applied according to 200 ml/mu by adding 50kg of water; the 10% validamycin aqua is diluted by 600 times for application. The medicine is administered at the beginning of sporadic scab, and is administered 3 times every 5-7 days. The medication method comprises the following steps: spraying, and spraying on the front and back surfaces of the leaf.
Each cell was investigated for all leaves on 15 tobacco plants before each application and 10 days after the last application, and 9-stage investigation was conducted on leaves according to "tobacco pest classification and investigation method" (GBT 23222-2008). The disease rate, disease index and relative prevention effect of each treatment are counted, wherein the disease index =100 × ∑ (number of diseased leaves at each level × representative value at each level)/(total number of examined leaves × representative value at the highest level), and the relative prevention effect (%) = (control disease index-treatment disease index)/control disease index × 100.
The results of field plot experiment survey show that the morbidity and disease index of a control group (CK), 80% mancozeb wettable powder, 10% validamycin aqua and a GT-6 fermentation liquor treatment group are gradually increased along with the prolonging of the pesticide application time, the highest morbidity and disease index are achieved in the last survey, the morbidity is respectively 61.32%, 36.67%, 27.08% and 16.58%, the morbidity and disease index of the GT-6 fermentation liquor treatment group are obviously lower, and the highest prevention effect on the tobacco target spot can reach 71.26%.
Control effect of surface GT-6 on tobacco target spot
In the present specification, the embodiments are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Sequence listing
<110> Hunan agriculture university
Central South agricultural experimental station of China Tobacco
<120> a strain for antagonizing pathogenic bacteria of tobacco target leaf spot
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
agagtttgat cctggctcag 20
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctacggctac cttgttacga 20
Claims (2)
1. The strain for antagonizing pathogenic bacteria of the tobacco target spot is characterized in that the strain is pseudomonas aeruginosa GT-6, is preserved in China center for type culture collection, has the preservation date of 2021 year, 7 months and 30 days, and has the preservation number of CCTCC NO: m2021953, the preservation address is China, wuhan university, the taxonomy is named as Pseudomonas aeruginosa GT-6.
2. The use of a strain that antagonizes pathogenic bacteria of tobacco target spot disease as defined in claim 1 for controlling tobacco target spot disease.
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| CN111575199A (en) * | 2020-04-21 | 2020-08-25 | 华南农业大学 | Pseudomonas aeruginosa JT86 and application thereof in preventing and treating sclerotinia rot |
| CN112442548A (en) * | 2020-12-17 | 2021-03-05 | 湖南省烟草公司郴州市公司 | LAMP primer group containing kit for detecting tobacco target spot disease, application and detection method thereof |
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| CN111575199A (en) * | 2020-04-21 | 2020-08-25 | 华南农业大学 | Pseudomonas aeruginosa JT86 and application thereof in preventing and treating sclerotinia rot |
| CN112442548A (en) * | 2020-12-17 | 2021-03-05 | 湖南省烟草公司郴州市公司 | LAMP primer group containing kit for detecting tobacco target spot disease, application and detection method thereof |
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