CN112210503A - Bacterial strain of non-pathogenic ralstonia solanacearum transformed with bacteriophage trp574 gene and preparation method and application thereof - Google Patents
Bacterial strain of non-pathogenic ralstonia solanacearum transformed with bacteriophage trp574 gene and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a non-pathogenic ralstonia solanacearum strain for transforming a phage trp574 gene, and a preparation method and application thereof. The strain is preserved in Guangdong province microorganism culture collection center in 2020, 7, 15, with the preservation number of GDMCC No. 61049. According to the invention, by adopting a molecular biology technology, the gene trp574 of the phage is introduced into the tobacco bacterial wilt to obtain the transgenic bacterial wilt engineering bacteria, and the engineering bacteria lose pathogenicity, but have stronger colonization ability in rhizosphere soil and tobacco plant bodies and show the prevention and treatment effect on the tobacco bacterial wilt.
Description
Technical Field
The invention relates to the technical field of ralstonia solanacearum prevention and control, and particularly relates to a pathogenicity-free ralstonia solanacearum strain transformed with a phage trp574 gene, and a preparation method and application thereof.
Background
Tobacco bacterial wilt caused by Laurella solanacearum (commonly called as Ralstonia solanacearum) is a destructive disease in tobacco production, and brings huge economic loss to tobacco industry in Yangtze river basin and areas in south of China. The ralstonia solanacearum has wide host range, favors high temperature, has complex population and is very difficult to control. The production mainly comprises agricultural control, disease-resistant breeding and chemical control, but the effect is not ideal. The method for preventing and treating bacterial wilt by using disease-resistant varieties is an economic and easy-to-operate measure, however, high-quality and high-yield and high-resistance tobacco varieties are still lacking in China at present, and the disease-resistant varieties gradually lose resistance in the long-term planting process due to environmental changes or variation of pathogenic bacteria. The rice-tobacco rotation mode is adopted, the limitation of the farming system is also applied in some places, and the tobacco can be planted in dry land, and the rotation period can reach more than three years to obtain better effect. The chemical control of bacterial wilt is difficult, because no ideal chemical agent exists at present, agricultural streptomycin which is applied all the time has already exited the market, the application of other chemical agents, such as copper agents of dragon bacterium, copper oxychloride, copper sulfate and the like, has a certain effect of delaying and slowing down the occurrence of bacterial wilt, but the control problem of bacterial wilt cannot be fundamentally solved, and the application of a large amount of chemical agents can cause the generation of drug resistance of pathogenic bacteria and environmental pollution, so the biological control of bacterial wilt is widely regarded at home and abroad.
Wherein, the use of the bacterial strain without pathogenicity for preventing and treating the bacterial wilt of crops is one of the key points of the research in the biological prevention and treatment of the bacterial wilt at present, in the prior art (Dongchun, Zengximing, Liu Jong, research for preventing and treating the bacterial wilt without pathogenicity by the bacterial strain without pathogenicity [ J ] the university of south China college of agriculture, 1999,020(004), 1-4, Chengqing river, prosperous and Hufangping, the prevention and treatment effect of the bacterial strain without pathogenicity on the bacterial wilt of tomato [ J ] the biological prevention and treatment of China, 2004,20(1):42-44.) although there is a report that the bacterial strain without pathogenicity is successfully used for preventing and treating the bacterial wilt, however, the control effect still needs to be improved, and in the prior art, the ralstonia solanacearum pathopoiesia strain is obtained mainly by adopting ultraviolet mutagenesis, plant tissue separation, pathogenic gene knockout and other modes, and no relevant report that exogenous genes are transferred into ralstonia solanacearum to obtain the ralstonia solanacearum is found.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a bacterial strain without virulence, which is transformed into a phage trp574 gene.
Another object of the present invention is to provide a method for producing the nonpathogenic ralstonia solanacearum strain of the transgenic phage trp574 gene.
The invention also aims to provide application of the transgenic phage trp574 gene.
The above object of the present invention is achieved by the following technical solutions:
a bacterial strain Ralstonia solanacearum TRP574 transformed with bacteriophage TRP574 gene is characterized in that the bacterial strain is preserved in Guangdong province collection center of microorganism strains in 7-15 months in 2020, with the preservation number being GDMCC No. 61049, the preservation unit address: lou 59 of Guangdong institute for microorganisms, Michelia Toxico 100, Guangzhou, Guangdong province, China.
According to the invention, a bacteriophage trp574 gene is introduced into tobacco bacterial wilt through a molecular biology technology to obtain transgenic bacterial wilt engineering bacteria, the engineering bacteria lose pathogenicity on bacterial wilt, but have strong colonization ability in rhizosphere soil and tobacco plant bodies, and show prevention and control effects on tobacco bacterial wilt.
The invention also protects the preparation method of the avirulent ralstonia solanacearum strain, which is to transform the trp574 gene of the phage into the ralstonia solanacearum strain to obtain genetically engineered bacteria; the nucleotide sequence of the phage trp574 gene is shown in SEQ ID NO:1 is shown.
Preferably, the recombinant expression vector containing trp574 gene is transformed into ralstonia solanacearum strain to obtain genetically engineered bacterium.
Preferably, the expression vector is pBBR1MCS 4.
Preferably, the ralstonia solanacearum is Tb 1546.
The invention also provides application of the pathogenic-free ralstonia solanacearum strain in controlling bacterial wilt or in preparing a bacterial wilt control medicament.
The invention also provides a method for preventing and treating bacterial wilt, which is to spray the bacterial liquid of the bacterial strain without pathogenicity around the roots of bacterial wilt crops.
Preferably, the concentration of the bacterial liquid is 2 x 106～5×106CFU/mL。
The invention also provides a medicament for preventing and treating bacterial wilt, which contains the nonpathogenic pseudomonas solanacearum.
Also provides application of the bacterial wilt prevention and control medicament in bacterial wilt prevention and control.
Preferably, the bacterial wilt is tobacco bacterial wilt.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a bacterial strain Ralstonia solanacearum TRP574 transformed with a bacteriophage TRP574 gene, which is preserved in Guangdong province collection center of microorganism strains in 7-15 months in 2020 with the preservation number of GDMCC No. 61049. According to the invention, by adopting a molecular biology technology, the phage trp574 gene is introduced into the tobacco bacterial wilt, so that the obtained genetic engineering bacteria lose the pathogenicity to the bacterial wilt, but have strong colonization ability in rhizosphere soil and tobacco plant bodies, show the prevention and treatment effect on the tobacco bacterial wilt, and have the prevention and treatment effect of 65.18%.
Drawings
FIG. 1 shows the colony morphology differences of Tb1546 and its trans-orf 30 gene strains. A. B, C are Tb1546, orf30-Tb1546, pBBR-Tb1546, respectively.
FIG. 2 shows the index change of tobacco bacterial wilt disease after Tb1546 and orf30-Tb1546 were inoculated by needle punching and irrigation. Note: a and B are the morbidity after Tb1546 and orf30-Tb1546 are inoculated by a needle punching method, and E is the disease index statistics; c and D are the disease conditions after Tb1546 and orf30-Tb1546 are inoculated by irrigation, and F is the statistics of disease index.
FIG. 3 shows the control effect of 30d potted plants after the engineering bacteria orf30-Tb1546 is applied.
FIG. 4 shows the incidence and disease index of tobacco bacterial wilt disease at 60d after the application of engineering bacteria orf30-Tb 1546.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
LB liquid medium: 5g of yeast extract, 10g of sodium chloride and 10g of tryptone, and distilled water is added until the volume is 1000 mL.
LB solid medium: 10g of tryptone, 5g of yeast extract, 10g of NaCl, 15g of agar powder and 1000mL of distilled water.
TTC medium: hydrolyzed casein 1g, peptone 10g, glycerol 5mL, agar 32g, distilled water 1000mL, 1% triphenyltetrazolium (TTC)10mL before use.
NA medium: 3g/L of beef extract, 5g/L of peptone, 0.5g/L of yeast extract, 10 g/L of glucose, 15g/L of agar powder and 7.0 of pH.
And (3) sowing the tested tobacco variety K326 in a hole pot, transplanting the tobacco seedling to a nutrition cup after the tobacco seedling grows 2 leaves, and using the tobacco seedling for pot culture test when the tobacco seedling is 5-6 leaves old.
The ralstonia solanacearum strain Tb1546 to be tested is a strong pathogenic strain, is separated from a tobacco producing area in Jian city, Jiangxi province, and is stored in a plant bacteria research room of southern China agricultural university.
Plasmid pBBR1MCS4 was stored in the plant bacteria research laboratory of southern China university of agriculture.
EXAMPLE 1 preparation of bacterial wilt-resistant engineered bacterium transformed with bacteriophage trp574 gene
1. Construction of recombinant plasmids
The inventor discovers a transcription regulatory factor orf30 by carrying out genome sequencing on lytic phage P574, further designs an amplification primer of a target gene orf30 according to P574 genome information, takes phage DNA as a template, carries out PCR amplification by taking the phage DNA as the template to obtain a target fragment of about 688bp, and carries out sequencing on the target fragment to discover that the orf30 sequence is 555bp, the nucleotide sequence of the orf30 sequence is shown as SEQ ID NO. 1, and the coded amino acid sequence is shown as SEQ ID NO. 2; the BALST alignment search found that orf30 has 82% homology with Ralstonia phase GP4, and the protein search results showed that it has a conserved sequence of transcription regulatory factor (transcription regulator), and the amino acid sequence homology was not higher than 70%. Therefore, rf30 was named as trp574 gene, where T stands for Transcriptional regulator, R stands for Ralstonia (Ralstonia), and P574 stands for this gene from bacteriophage P574.
Further, a target fragment of rf30(trp574 gene) was transferred to plasmid pBBR1MCS4 by one-step cloning. plasmid extraction of pBBR1MCS4 was performed according to the Axygen plasmid miniprep kit, and after double digestion with BamHI and Hind III, Novozam was used
II One Step Cloning Kit ligation and heat shock transformation to DH5 α. The plated plates were cultured in an inverted state at 37 ℃ for 24 hours until single colonies appeared, and colony PCR was performed using MCS-F/MCS-R primers (shown in Table 1) in the following PCR system: template 5. mu.L, 10 XBuffer 1. mu.L, dNTP 0.8. mu.L, MSC-F0.4. mu.L, MSC-R0.4. mu.L, Taq enzyme 0.08. mu.L, ddH2O2.32. mu.L, 10. mu.L in total. The PCR procedure was: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 40 s; 72 ℃ for 5min, 16 ℃. Liquid culture is carried out on the bacterial strain containing the target band, plasmids are extracted and enzyme digestion verification is carried out by using BamHI and Hind III, and meanwhile, the recombinant plasmids are handed over to the Yinxie substrate for sequencing and identification. The obtained recombinant plasmid was named pBBR-orf 30.
TABLE 1 orf30-F/orf30-R and MCS-F/MCS-R primer sequences
The recombinant plasmid pBBR-orf30 is successfully constructed after enzyme digestion and sequencing.
2. Preparation and electrotransformation of ralstonia solanacearum competent cells
(1) The ralstonia solanacearum Tb1546 is inoculated in 10mL of NA culture medium for overnight culture, the bacterial liquid is placed on ice for 15min, the bacterial liquid is subpackaged into precooled 1.5mL centrifuge tubes, the centrifugal operation is carried out at 4 ℃ and 6000rpm for 5min, and the supernatant is discarded.
(2) 1mL of 10% glycerol was added for resuspension, and the mixture was centrifuged at 6000rpm at 4 ℃ for 5min, and the supernatant was discarded.
(3) And (3) repeating the step (2) twice.
(4) Add 10% glycerol 100 u L heavy suspension obtained Ralstonia solanacearum competence.
(5) 50 mu L of prepared Tb1546 ralstonia is taken to be competent in a 0.1cm electric shock cup, about 2 mu g of recombinant plasmid pBBR-orf30 is added, and the voltage is set to be 1.4kv in a manual mode for electric shock transformation. Meanwhile, about 2 mu g of pBBR1MCS4 plasmid is taken to carry out empty vector electric shock transformation in the pseudomonas solanacearum competence. Adding 800 mu L of NA liquid culture medium to recover for 12h immediately after transformation is finished, coating a plate on the NA culture medium containing Amp antibiotics, carrying out PCR screening on transformants orf30-Tb1546 and pBBR-Tb1546, and constructing PCR primers and recombinant plasmids in the same condition step.
Successfully transforming the recombinant plasmid into ralstonia solanacearum Tb1546 by electric shock to obtain a transgenic strain orf30-Tb 1546; meanwhile, the empty plasmid is transferred into Tb1546 by electric shock to obtain pBBR-Tb 1546. The ralstonia solanacearum can form pink colonies on a TTC culture medium, a large amount of white extracellular polysaccharide is generated outside the pink colonies, and the stronger the pathogenicity is, the higher the extracellular polysaccharide content is. Tb1546 and pBBR-Tb1546 were able to form smooth pink colonies and produce large amounts of extracellular polysaccharide on TTC, while orf30-Tb1546 strain transformed with orf30 gene formed shriveled colonies on TTC plates with less extracellular polysaccharide content, indicating reduced virulence (FIG. 1).
3. orf30-Tb1546 bacterial wilt engineering bacterium pathogenicity determination
The tobacco K326 was cultivated in pot plants until the seedling stage, and 20 plants were used as a group. Tb1546 and orf30-Tb1546 were cultured in NA until logarithmic growth phase, and the concentration of the culture was adjusted to about 1X 108cfu/mL, 1mL is extracted by an injector to stab and inoculate the tobacco stems in the seedling stage of the potted plant, cotton is covered on the wound, and redundant bacterial liquid is injected into the cotton. Within 3d after inoculation, the cotton is subjected to moisture preservation treatment every 12 h. Simultaneously, another batch of potted seedling stage tobacco is taken and irrigated by adopting an irrigation methodAnd (4) inoculating. Firstly, the root of the tobacco is scratched by a cutter, and the concentration of the bacteria is about 108The bacterial liquid of cfu/mL is diluted by 25 times, and each plant is irrigated by 500mL to ensure that the soil in the potted plant is thoroughly irrigated.
The number of disease progression is recorded every 2d from the onset of wild flora, and the disease index is counted. The disease progression refers to tobacco pest grading standard (GB/T23222 and 2008):
level 0: the whole plant is disease-free;
level 1: occasionally, the stem has chlorotic streak; or withering of leaves below the diseased side 1/2;
and 3, level: 1/2 with black streak but no higher than stem height; or withering of diseased side 1/2 to 2/3 leaves;
and 5, stage: stem black streaks exceed the stem height 1/2, but do not reach the top of the stem; or withering leaves above the diseased side 2/3;
and 7, stage: the black streak of the stem reaches the top of the stem; or withering all the leaves of the diseased plant;
and 9, stage: the diseased plants die basically.
The disease index formula is: disease index (Σ (each representative value × number of plants) ÷ highest representative value × total number of plants) × 100.
The result shows that the infection effect of pathogenic bacteria can be more obviously displayed by the needle punching method. After onset, the index of the tobacco bacterial wilt treated by the needle punching method is obviously faster than that of the tobacco wilt treated by the irrigation method (figure 2). The needling inoculation result shows that the control group inoculated with Tb1546 is attacked 22d later, the experimental group inoculated with orf30-Tb1546 is attacked, and the attack rate is stable after 34 d. At this time, the difference between the disease indexes of the control group and the experimental group is more than 50. The irrigation experiment group does not attack the disease at all, and the final disease index of the control group reaches more than 40. The results show that the pathogenicity of orf30-Tb1546 to tobacco is obviously reduced, orf30 obviously influences the pathogenicity of Tb1546, and the invention obtains the non-pathogenic bacterial strain engineering by transferring phage trp574 gene by transferring phage exogenous gene orf30(trp574) into a ralstonia solanacearum strain. The obtained orf30-Tb1546 genetically engineered bacterium was named Ralstonia solanacearum TRP574 and was deposited in Guangdong province culture Collection (GDMCC) at 7/15 th 2020, address: building 5, lou 59, institute for microbiology, guangdong province, code, junior 100, maeli, guangzhou city: 510070, accession number: GDMCC No: 61049.
example 2 potting test for tobacco bacterial wilt control
1. Method of producing a composite material
Culturing ralstonia solanacearum: activating Tb1546 stored at-80 deg.C with TTC culture medium, culturing at 30 deg.C for 48h to obtain bacterial colony of ralstonia solanacearum, and selecting single bacterial colony to culture in LB liquid culture medium for 24 h. Similarly, the activated orf30-Tb1546 strain is cultured for 24-48 h by LB liquid for standby. And (3) pot culture inoculation: time 2019, 6 and 26 months, was carried out in a south china university of agriculture, bacteria research laboratory greenhouse. Each pot is filled with 15kg of soil, 2 tobacco seedlings with 6 leaf ages are planted, the number of each tobacco seedling is 48, and the pot number is 24. Inoculating engineering bacteria orf30-Tb1546 strain, inoculating 200mL of engineering bacteria liquid with concentration of 2X 106CFU/mL. The next day, the concentration was 5X 106CFU/mL ralstonia solanacearum Tb1546 is sprayed around tobacco roots, cross-shaped soil at the roots of tobacco seedlings is scratched by a knife, the tobacco seedlings are wounded with roots, 200mL of bacterial strain is sprayed in each pot, engineering bacteria are inoculated every 15d for 3 times, and the control group (CK) which is inoculated with Tb1546 and 3 times of clear water is inoculated. And (3) placing the potted tobacco after inoculation in a greenhouse, keeping the temperature at 25-30 ℃, watering regularly, and performing conventional management.
The incidence and disease index of bacterial wilt are regularly investigated. And calculating disease index and prevention and treatment effect of each treatment 2 months after inoculation. The disease index grading standard and the control effect are calculated as follows:
bacterial wilt investigation grading standard (taking the whole plant as a unit)
Tobacco bacterial wilt disease grading standard GB/T23222-
Level 0: the tobacco plant is disease-free;
level 1: occasionally, the stem has chlorotic streak; or withering of leaves below the diseased side 1/2;
and 3, level: the stem has black streak spots, but does not exceed the stem height 1/2; or withering of diseased side 1/2 to 2/3 leaves;
and 5, stage: stem black streaks exceed stem height 1/2, but do not reach the top of the stem; or withering leaves above the diseased side 2/3;
and 7, stage: the black streak of the stem reaches the top of the stem; or withering all the leaves of the diseased plant;
and 9, stage: the diseased plant dies basically
Control effect [ (incidence of control-incidence of treatment ]/incidence of control × 100%. Disease index ∑ number of symptoms at each level × number of plants/(highest symptom representative × total number of plants) × 100
2. Results
After potted tobacco is planted for one week, CK treatment starts to attack, and engineering bacteria orf30-Tb1546 treatment does not attack, until 15d, bacterial wilt disease strains begin to appear, which shows that the engineering bacteria have the function of delaying bacterial wilt attack, the early-stage control effect is obvious (figure 3), the incidence rate of the bacterial wilt gradually increases along with the time, and the disease grade number of each treatment is increased by 2 months after inoculation (Table 2).
TABLE 2 incidence of the cultivated tobacco 60 days after application of the engineered bacterium orf30-Tb1546
| Grading of disease conditions | Number of CK-developing plants | orf30-Tb1546 number of |
| 0 | 5 | 25 |
| 1 | 2 | 5 |
| 3 | 4 | 6 |
| 5 | 6 | 4 |
| 7 | 5 | 3 |
| 9 | 26 | 5 |
According to the investigation of 28 days 8 and 8 months in 2019, the control bacterial wilt morbidity rate is 89.58%, the disease index is 72.45, the engineering bacteria treatment morbidity rate is 47.92%, the disease index is 25.23 (shown in figure 4), and the prevention and treatment effect on the tobacco bacterial wilt is 65.18% by applying the engineering bacteria orf30-Tb1546 for treatment according to the calculation of the disease index. The bacterial wilt-free engineering bacterium transformed with the trp574 gene can be used for preventing and treating tobacco bacterial wilt, and has a wide application prospect.
Sequence listing
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Claims (10)
1. A bacterial strain of ralstonia solanacearum transformed with a phage trp574 gene is characterized in that the bacterial strain is preserved in Guangdong province microorganism culture collection center at 7-15 th month in 2020, and the preservation number is GDMCC No. 61049.
2. The method for producing a. avirulent ralstonia strain according to claim 1, wherein a gene of bacteriophage trp574 is transformed into a. avirulent strain to obtain a genetically engineered bacterium; the nucleotide sequence of the phage trp574 gene is shown in SEQ ID NO:1 is shown.
3. The method of claim 2, wherein the genetically engineered bacterium is obtained by transforming a recombinant expression vector containing the trp574 gene into a ralstonia solanacearum strain.
4. Use according to claim 2 or 3, wherein the ralstonia solanacearum is Tb 1546.
5. The use of the avirulent ralstonia solanacearum strain of claim 1 for controlling bacterial wilt or for preparing a drug for controlling bacterial wilt.
6. A method for controlling bacterial wilt, characterized in that the bacterial liquid of the avirulent ralstonia strain of claim 1 is sprayed around the roots of a bacterial wilt crop.
7. The method according to claim 6, wherein the bacterial liquid concentration is 2 x 106～5×106CFU/mL。
8. An agent for controlling bacterial wilt, characterized by comprising the avirulent ralstonia strain according to claim 1.
9. The use of the agent of claim 8 for controlling bacterial wilt.
10. The use of claim 5 or 10, wherein the bacterial wilt is tobacco bacterial wilt.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410825A (en) * | 2018-04-20 | 2018-08-17 | 南京农业大学 | A kind of bacteriophage cocktail and its application |
| CN112029789A (en) * | 2020-08-03 | 2020-12-04 | 华南农业大学 | Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108410825A (en) * | 2018-04-20 | 2018-08-17 | 南京农业大学 | A kind of bacteriophage cocktail and its application |
| CN108410825B (en) * | 2018-04-20 | 2022-09-20 | 南京农业大学 | Phage cocktail and application thereof |
| CN112029789A (en) * | 2020-08-03 | 2020-12-04 | 华南农业大学 | Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage |
| CN112048514A (en) * | 2020-08-03 | 2020-12-08 | 华南农业大学 | Phage trp574 gene and application thereof |
| CN112029789B (en) * | 2020-08-03 | 2022-05-31 | 华南农业大学 | Application of phage trp574 gene in reducing resistance of ralstonia solanacearum to phage |
| CN112048514B (en) * | 2020-08-03 | 2022-08-23 | 华南农业大学 | Phage trp574 gene and application thereof |
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