CN112300975B - Low-pathogenicity mutant strain of pogostemon cablin ralstonia solanacearum and application thereof - Google Patents
Low-pathogenicity mutant strain of pogostemon cablin ralstonia solanacearum and application thereof Download PDFInfo
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Abstract
The invention discloses a low-pathogenicity mutant strain of pogostemon cablin ralston solanacearum and application thereof, belonging to the technical field of biological control of plant diseases. The invention takes a strong pathogenic strain Kosakonia sp.Pa82 of patchouli bacterial wilt as a material, and obtains a low pathogenic mutant strain Pa82-87-1 through Tn5 transposon insertional mutation. The growth environment and the nutritional requirements of Pa82-87-1 are the same as those of pathogenic bacteria Pa82, the Pa82-87-1 can compete with ralstonia solanacearum for survival sites and nutrients, and the potential disease resistance of plants is induced to play a role of plant vaccines, so that the infection and the propagation of the pathogenic bacteria are inhibited or slowed down. The low-pathogenicity mutant strain Pa82-87-1 is used as a test strain to carry out biological control tests on the patchouli bacterial wilt, and the result shows that the control effect of the mutant strain on the patchouli bacterial wilt can reach 71.70 percent and the mutant strain can be used as a biological control strain for controlling the patchouli bacterial wilt.
Description
Technical Field
The invention belongs to the technical field of biological control of plant diseases, and particularly relates to a low-pathogenicity mutant strain of ralstonia solanacearum and application thereof.
Background
Pogostemon cablin (Blanco) Benth, a plant of the genus Pogostemon of the family Labiatae, is used as a dry aerial part of a drug, and is one of the famous-region medicinal materials and the ten-large Guangdong medicinal materials. Patchouli faces severe bacterial wilt in production. Bacterial wilt is a vascular bundle disease caused by bacteria, can occur in the whole growth period of plants, and is most prevalent in high-temperature rainy seasons. The stem and leaf of the disease-affected plant will wither and droop until all the plants die. Once the patchouli plants get ill, the disease can spread rapidly, leading to the death of large-area plants and causing destructive loss.
At present, bacterial wilt is mainly chemically controlled. Since pathogenic bacteria can live in the soil, spraying chemical agents can only kill bacteria on the surface of the soil layer. The repeated use of the pesticide can easily cause the bacteria to generate drug resistance and cause pesticide residue in the medicinal materials and environmental pollution. In recent years, biological control plays an important role in the control of soil-borne diseases of crops. Antagonistic bacteria are widely studied due to strong inhibition on pathogenic bacteria, but studies show that the antagonistic bacteria screened indoors are unstable in prevention and treatment effects in fields due to the fact that the antagonistic bacteria do not adapt to ecological environments. The non-pathogenic or weak pathogenic strain is weakened or mutated by pathogenic bacteria, has the same growth environment and nutritional requirements as the pathogenic bacteria, can compete with the pathogenic bacteria and induce the immune response of plants, and can prevent and treat diseases by playing the role of plant vaccine.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a low-pathogenicity mutant strain of pogostemon velutina.
The invention also aims to provide application of the low-pathogenicity mutant strain of the pogostemon velutina.
The invention takes a highly pathogenic bacterial strain Kosakonia sp.Pa82 of patchouli bacterial wilt as a material, and performs Tn5 transposon insertion mutation on the strain by an electric shock transformation method to obtain a mutant. The mutant strain Pa82-87-1 with weakened pathogenicity to patchouli is obtained by screening pathogenicity of a large number of mutants. The Tn5 transposon insertion site gene of the low pathogenicity mutant strain Pa82-87-1 is cloned, and the bioinformatics analysis result shows that the coding protein of the transposon insertion site gene of the mutant strain Pa82-87-1 is a type VI secretion system protein VgrG. The low-pathogenicity mutant strain Pa82-87-1 is used as a test strain to carry out biological control test on the patchouli bacterial wilt, and the result shows that the control effect of the mutant strain on the patchouli bacterial wilt can reach more than 70 percent (71.70 percent).
The purpose of the invention is realized by the following technical scheme:
the invention provides a low-pathogenicity mutant strain of patchouli bacterial wilt, which is named as Kosakonia sp.Pa82-87-1 and is obtained by taking a strong-pathogenicity pathogenic strain of patchouli bacterial wilt Kosakonia sp.Pa82 as a material and performing insertion mutation by Tn5 transposon.
The deposit information of the strain Kosakonia sp.Pa82-87-1: the preservation unit: guangdong province microbial culture Collection (GDMCC), accession number: GDMCC No: 61048, preservation address: the microbiological research institute of Guangdong province, No. 59 building, No. 5 building, Guangdong province, of the Fuli Zhonglu 100, Guangzhou city, the preservation date: 2020, 7, 16 months.
The Tn5 transposon insertion mutant gene of the low virulence mutant Pa82-87-1 is cloned, and the bioinformatics analysis result shows that the protein coded by the transposon insertion mutant gene of the mutant Pa82-87-1 is a VI type secretion system protein VgrG.
And (3) carrying out a test for preventing and treating the patchouli bacterial wilt on the low-pathogenicity mutant strain Pa 82-87-1. The plant which is pre-inoculated with the low-pathogenicity mutant strain Pa82-87-1 and is inoculated with the wild strain Pa82 is taken as a biocontrol group, the plant which is pretreated with water and is inoculated with the wild strain Pa82 is taken as a positive control group, and the plant which is only treated with water is taken as a negative control group, so that the control effect of the mutant strain on the patchouli bacterial wilt can reach 71.70 percent.
The low-pathogenicity mutant strain of the patchouli bacterial wilt is applied as a biocontrol strain of the patchouli bacterial wilt.
The low-pathogenicity mutant strain of the pogostemon cablin bacterial wilt is applied to the preparation of plant vaccines for preventing and treating bacterial wilt.
A plant vaccine for preventing and treating bacterial wilt is prepared from the low-pathogenicity mutant of Pogostemon cablin bacterial wilt.
The plant vaccine for preventing and treating bacterial wilt has the concentration of about 4.5 multiplied by 108～5.5×108cfu/mL (preferably 5X 10)8cfu/mL) of the low virulence mutant strain Pa 82-87-1.
The preparation method of the plant vaccine for preventing and treating the bacterial wilt comprises the following steps:
inoculating low-pathogenicity mutant strain Pa82-87-1 into NA liquid culture medium, performing constant temperature shaking culture at 28 deg.C and 200r/min for 24h, diluting with sterile water 2.5 times, and making the bacterial liquid concentration be 4.5 × 108～5.5×108cfu/mL (preferably 5X 10)8cfu/mL) to obtain the plant vaccine for preventing and treating bacterial wilt.
The plant vaccine for preventing and treating the bacterial wilt is applied to preventing and treating the patchouli bacterial wilt.
Compared with the prior art, the invention has the following advantages and effects:
(1) the bio-control strain Pa82-87-1 is a low-pathogenicity mutant strain obtained by inserting and mutating a strong-pathogenicity pathogenic strain Kosakonia sp.Pa82 of patchouli bacterial wilt through Tn5 transposon, the growth environment and the nutritional requirements of the low-pathogenicity mutant strain are the same as those of pathogenic bacteria, the low-pathogenicity mutant strain can compete with ralstonia solanacearum for survival sites and nutrients, and the action of a plant vaccine is exerted by inducing the potential disease resistance of plants, so that the infection and the propagation of the pathogenic bacteria are inhibited or slowed down.
(2) Greenhouse experiments show that the biocontrol strain can reduce the occurrence of patchouli bacterial wilt. The low-pathogenicity mutant strain Pa82-87-1 is taken as a test strain, bacterial liquid cultured for 24 hours is taken and diluted by 2.5 times by sterile water, and a biological control test is carried out on the patchouli plants, and the result shows that the mutant strain has the control effect on the patchouli bacterial wilt reaching 71.70 percent and can be used as a biological control strain for controlling the patchouli bacterial wilt.
Drawings
FIG. 1 is Kan of resistant clonesrPCR identification of the gene; wherein M is a 2000bp DNA Marker; p is Tn5 transposon (positive control); WT is wild strain Pa82 (negative control); 1-12 are resistant clones.
FIG. 2 is the pathogenicity of the mutant strain Pa82-87-1 to Pogostemon cablin plant.
FIG. 3 is an amplification of sequences flanking the transposon insertion site of the low virulence mutant Pa 82-87-1; wherein M is a DNA Marker of 2000 bp; 1 is mutant strain Pa 82-87-1; 2 is wild strain Pa 82.
FIG. 4 is a PCR amplification of transposon insertion site gene of low virulence mutant Pa 82-87-1; wherein M is a DNA Marker of 5000 bp; 1 is wild strain Pa 82; 2 is mutant strain Pa 82-87-1.
FIG. 5 is a double restriction enzyme identification of recombinant plasmids; wherein M is a DNA Marker of 5000 bp; 1 is the Nhe I single enzyme digestion result; 2 is the result of double enzyme digestion of Nhe I and Spe I.
FIG. 6 shows the control results of the low virulence mutant strain Pa82-87-1 on patchouli bacterial wilt.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
The strain Kosakonia sp.Pa82 used in the examples was isolated and purified from Pogostemon cablin plants having the symptoms of typical bacterial wilt; the preservation information is as follows: the preservation unit: guangdong province microbial culture Collection (GDMCC), accession number: GDMCC No: 61047, deposit address: the microbiological research institute of Guangdong province, No. 59 building, No. 5 building, Guangdong province, of the Fuli Zhonglu 100, Guangzhou city, the preservation date: year 2020, 7, 16.
Example 1 construction of transposon insertion mutant of Strain Kosakonia sp.Pa82 Tn5
Transposon kit EZ-Tn5TM＜KAN-2＞Tnp TransposomeTMKit, purchased from Epicentre, USA, is a Tn5 transposon (transposon) and transposase (transposase) complex with a 1221bp overall length, carrying kanamycin resistance in the middle (Kan)r) A gene.
Preparation of competent cell of Strain Kosakonia sp.Pa82 (see "Wang Yao, Zhang Yao, Hehong, etc.. Pogostemon Hibiscus Tn5 transposon insertion mutant construction [ J]J. Chinese materia medica, 2019,44(01):77-81. "), 100. mu.L of competent cell suspension of the strain Pa82 and 1. mu.L of Tn5 transposon are respectively sucked and fully mixed, transferred to a precooled electric shock cup with the length of 0.2cm, and placed on ice for 5 min. The electric shock conditions are as follows: voltage 2.5kV, capacitance 25 muf and resistance 400 omega. Rapidly adding 900 μ L SOC culture medium in 30 deg.C warm bath after electric shock, mixing, recovering and culturing at 30 deg.C for 1 hr at 200r/min, spreading recovering bacteria liquid on TTC solid culture medium containing 10mg/L kanamycin, and culturing at 30 deg.C for 24 hr in an inverted manner. Resistant clones were picked for KanrPCR amplification of the gene (P1: 5'-GGTGCGACAATCTATCGA-3' and P2: 5'-CTCATCGAGCATCAAATG-3') and electrophoresis detection results are shown in FIG. 1, and Tn5 transposon (positive control) and the resistant clone show specific bands at about 700bp, while the wild strain does not amplify the corresponding band, which indicates that Tn5 transposon insertion mutant of the strain Kosakonia sp.Pa82 is obtained by electric shock transformation.
Example 2 selection of Low-pathogenic mutant Strain Kosakonia sp.Pa82
Transposition of a series of strains Pa82 obtained in example 1The daughter insertion mutant strain was used as a test strain, and pathogenicity screening was performed with the wild strain Pa82 as a control. Respectively performing liquid culture on the strains, wherein the culture medium is NA liquid culture medium, performing constant temperature shaking culture at 28 deg.C and 200r/min for 24 hr, diluting with sterile water 2.5 times, and the concentration of bacterial liquid is about 5 × 108cfu/mL; selecting healthy patchouli plants with the seedling age of about 45 days, respectively inoculating diluted bacterial liquid to the patchouli plants by a root injury soaking method, and treating the patchouli plants with sterile water as a blank control group. And (3) transferring the plants to a greenhouse shed, maintaining the humidity at about 85% and the temperature at 28-30 ℃, and observing and recording the disease occurrence condition of the plants for 1-7 days. The experiment was repeated 3 times. Through pathogenicity screening, 1 mutant strain with obviously weakened pathogenicity to the cablin potchouli herb is obtained and marked as Pa82-87-1, and the pathogenicity of the strain Pa82-87-1 to the cablin potchouli herb is shown in figure 2. The disease index of the plant treated by sterile water is 0, the disease index of the plant inoculated with the strain Pa82 is the highest, and reaches more than 80% at the 7 th d of inoculation, and the disease index of the plant inoculated with the mutant strain Pa82-87-1 is obviously reduced compared with that of a wild strain. Through pathogenicity screening, a low-pathogenicity mutant strain Pa82-87-1 is obtained.
Example 3 cloning of transposon insertion site Gene of Low virulence mutant Pa82-87-1
(1) Amplification of sequences flanking the Tn5 transposon insertion site
Extracting genomic DNA of the low-virulence mutant Pa82-87-1, digesting the genomic DNA with restriction enzyme Hind III, and performing digestion with T4And (4) connecting the enzyme digestion products by using ligase. Primers KAN-2FP-1 (5'-ACCTACAACAAAGCTCTCATCAACC-3') and KAN-2RP-1 (5'-GCAATGTAACATCAG AGATTTTGAG-3') designed based on the transposon sequence of Tn5 were used to perform reverse PCR amplification of the ligation products to obtain the flanking sequences of the transposon insertion site of mutant Tn 5. Reaction system: ligation product 1.0. mu.L; primers (10. mu.M) were 1.0. mu.L each; 12.5 mu L of Taq premix enzyme; ddH2O9.5. mu.L. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 2min, and 35 cycles; extension at 72 ℃ for 7 min. The reverse PCR product was subjected to agarose gel electrophoresis detection, and the results are shown in FIG. 3, wherein 1 band was amplified from the low virulence mutant Pa82-87-1, and the negative control bacteriumThe strain Pa82 did not produce a desired band. And (4) sequencing the reverse PCR product to obtain the flanking sequences of the Tn5 transposon insertion site.
(2) Cloning and sequencing of Tn5 transposon insertion site gene
Designing a specific primer according to a homologous gene sequence obtained by aligning Tn5 transposon insertion site flanking sequences BLAST: 87-1-F: 5' -CTAGCTAGCATGTTGAACAGAATTACGGTCAAG-3' (Nhe I cleavage site underlined), 87-1-R: 5' -GGACTAGTTTACGGTTTCGACTTTTTAGCATC-3' (Spe I cleavage site underlined). Respectively taking the genomic DNA of the wild strain and the mutant strain as templates and taking 87-1-F and 87-1-R as primers to carry out PCR amplification on the target gene. Reaction system: 45 mu L of high-fidelity premixing enzyme; primers (10. mu.M) 2.0. mu.L each; DNA template 1.0. mu.L. Reaction conditions are as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 3 min.
And (3) carrying out agarose gel electrophoresis detection on the PCR product, wherein the result is shown in figure 4, specific bands are amplified by the wild strain Pa82 and the low-pathogenicity mutant strain Pa82-87-1, the size of the bands is consistent with the expected result, the total length of the transposon is 1221bp, and the bands of the mutant strain are about 1200bp longer than that of the wild strain. And (3) carrying out gel cutting recovery on a PCR amplification product of the Pa82 genome. Connecting the purified product with a T vector to construct a recombinant plasmid, transforming the recombinant plasmid into an escherichia coli competent cell by adopting a heat shock method, coating the transformed bacterial liquid on an LB solid culture medium containing ampicillin (100 mu g/mL), and culturing overnight. Selecting the resistant bacterial colony, carrying out PCR identification, further extracting positive clone plasmid, carrying out Nhe I and Spe I double enzyme digestion identification, wherein the enzyme digestion result is shown in figure 5, the sizes of the double enzyme digestion product fragments are respectively basically consistent with the sizes of the target gene and the vector, and the size of the single enzyme digestion product fragment is about the sum of the lengths of the target gene and the vector, which indicates that the construction of the recombinant plasmid is successful. Through sequencing, the nucleotide sequence of the transposon insertion site gene of the low virulence mutant Pa82-87-1 is shown as SEQ ID NO: 5, the total length is 2613bp, and the coded protein is VI type secretion system protein VgrG, Tn5 transposon is inserted between 2144bp and 2145bp of the gene.
The low pathogenicity mutant strain Pa82-87-1 is named as Kosakonia sp.Pa82-87-1, and the preservation information thereof is as follows: the preservation unit: guangdong province microorganism culture Collection (GDMCC), preservation number: GDMCC No: 61048, preservation address: the microbiological research institute of Guangdong province, No. 59 building of Michelia Tokyo No. 100 college, Guangzhou city, preservation date: year 2020, 7, 16.
Example 4 prevention of Low virulence mutant Pa82-87-1 against patchouli bacterial wilt
Using low-pathogenicity mutant strain Pa82-87-1 as test strain, inoculating the strain into NA liquid culture medium, performing constant temperature shaking culture at 28 deg.C and 200r/min for 24h, diluting with sterile water 2.5 times, and making the concentration of the bacterial liquid about 5 × 108cfu/mL. Healthy patchouli plants with the seedling age of about 45 days are taken as the material. The biocontrol group pre-inoculates a low-pathogenicity mutant strain Pa82-87-1 to the roots of the patchouli plants after root irrigation, and then damages the roots and soaks and inoculates a wild strain Pa82 after 3 days. And (3) irrigating the positive control group with sterile water to pre-treat the plants, and after 3d, damaging roots and soaking to inoculate the wild strain Pa 82. Negative controls plants were treated with water all the time. The test plants were transferred to an environment suitable for disease development (humidity 85%, temperature 28-30 ℃). And respectively recording the disease occurrence conditions of the plants inoculated for 1-7 d. The experiment was repeated 3 times. As shown in FIG. 6, after inoculation for 7d, the disease index of the positive control group is 73.61%, the disease index of the biocontrol group is 20.83%, and the control effect of the low pathogenicity mutant strain Pa82-87-1 on the patchouli bacterial wilt reaches 71.70%.
The disease grade of the plants is 5 grades: the 0 grade is plant health; grade 1 is less than 25% of plants with drooping leaves and wilting; 2-grade 26% -50% of plants with drooping leaves and wilting leaves; 3-grade plant with 51-75% of leaf droop and wilting; grade 4 is 76-100% of plants with leaf droop, wilting or stalk bending.
The disease index of the plant is [ ∑ (number of diseased plants at each level × corresponding number of levels)/(total number of tested plants × highest-level value) ] × 100%.
The preventing and treating effect (%) is (disease index of positive control group-disease index of biological control group)/disease index of positive control group x 100%.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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Claims (7)
1. A low pathogenicity mutant strain of pogostemon cablin ralstonia solanacearum is characterized in that: the name of the bacterium Coxsackii (Kosakoniasp.）Pa82-87-1, deposited at 16.7.2020 in the Guangdong province collection center for microbial cultures of Guangdong province, institute of microbiology, Guangdong province, No. 59 building, Guangdong province, institute of microbiology, Mieli Zhou 100, Guangzhou, with accession number: GDMCC No: 61048.
2. the use of the low-virulence mutant strain of pogostemon solanacearum of claim 1 as a biocontrol bacterium of pogostemon solanacearum, characterized in that:
the patchouli bacterial wilt is determined by a preservation number GDMCC No: 61047 Kosakholderia sp. (C.)Kosakoniasp.) Pa82 infection.
3. The use of the low-virulence mutant strain of pogostemon solanacearum of claim 1 in the preparation of a plant vaccine for preventing and treating pogostemon solanacearum, characterized in that:
the patchouli bacterial wilt is determined by a preservation number GDMCC No: 61047 Kosakholderia sp. (C.)Kosakoniasp.) Pa82 infection.
4. A plant vaccine for controlling bacterial wilt, characterized in that it is prepared by using low-pathogenicity mutant strain of Pogostemon cablin bacterial wilt of claim 1.
5. The plant vaccine for controlling bacterial wilt according to claim 4, wherein:
the plant vaccine for preventing and treating bacterial wilt contains the components with the concentration of 4.5 multiplied by 108～5.5×108 The cfu/mL low-virulence mutant strain Pa 82-87-1.
6. The method for preparing a plant vaccine for controlling bacterial wilt disease according to claim 4 or 5, characterized by comprising the steps of:
inoculating low-pathogenicity mutant strain Pa82-87-1 into NA liquid culture medium, performing constant temperature shaking culture at 28 deg.C and 200r/min for 24h, diluting with sterile water 2.5 times, and making the bacterial liquid concentration be 4.5 × 108～5.5×108 cfu/mL, and obtaining the plant vaccine for preventing and treating bacterial wilt.
7. The application of the plant vaccine for preventing and treating bacterial wilt disease of claim 4 or 5 in preventing and treating patchouli bacterial wilt disease is characterized in that:
the patchouli bacterial wilt is determined by a preservation number GDMCC No: 61047 Kosakholderia sp. (C.)Kosakoniasp.) Pa82 infection.
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