CN112226384B - Pathogenic strain causing patchouli bacterial wilt and application thereof - Google Patents

Abstract

The invention discloses a pathogenic strain causing patchouli bacterial wilt and application thereof, belonging to the technical field of plant pathogenic bacteria detection. The pathogenic strain is Kosakonia sp.Pa82 with a preservation number: GDMCC No: 61047, is a new pathogenic strain of patchouli bacterial wilt. The pathogenic strain has strong pathogenicity on the patchouli, provides a research material for the research of related genes of bacterial wilt virulence and pathogenic mechanisms, and provides a theoretical basis for the prevention and treatment research of bacterial wilt diseases; the method has important significance for screening of the patchouli disease-resistant germplasm, breeding of disease-resistant varieties and research and development of bacterial wilt prevention and control plant vaccines, and provides a research idea for prevention and control research of other plant bacterial wilt.

Description

Pathogenic strain causing patchouli bacterial wilt and application thereof

Technical Field

The invention belongs to the technical field of plant pathogenic bacteria detection, and particularly relates to a pathogenic strain causing patchouli bacterial wilt and application thereof.

Background

Patchouli bacterial wilt is a vascular bundle disease caused by bacteria. Pathogenic bacteria invade from the root of the plant, colonize and multiply in vascular bundles, cause difficulty in water transportation of the plant, and cause wilting of stems and leaves until the plant is withered and dead. Pathogenic bacteria can live through the winter in soil and diseased and disabled tissues, and the disease attack on continuous cropping land is more serious. Bacterial wilt can occur in the whole growing period of plants, and the disease is most prevalent in high-temperature rainy seasons. Once plants are attacked, the disease spreads rapidly, and large areas of plants die. Bacterial wilt is a devastating disease in the production of patchouli. The separation and identification of pathogenic bacteria are important for preventing and treating patchouli bacterial wilt.

Bacterial wilt is one of the most serious plant diseases, and more than 50 plants of 450 families suffer from bacterial wilt, including medicinal plants such as pogostemon cablin, casuarina equisetifolia, momordica grosvenori and the like. The bacterial wilt pathogen first reported was Laurella solanacearum, which was first isolated from a plant of the family Solanaceae and is also called Laurella solanacearum (abbreviated as Ralstonia solanacearum). It has long been known that ralstonia solanacearum belongs to a complex species. Usually, the DNA-DNA homology in bacterial species should be up to 70%, but less than 70% in Ralstonia solanacearum. With the continuous and deep research of pathogenic bacteria of plants and the application of various molecular biological means, the original strains which have genetic relationship with each other and are classified as composite strains of ralstonia solanacearum are separated and classified into different genera. Some strains such as Erwinia, Kosakonia, etc., all reported to be pathogenic for bacterial wilt in different host plants, have similarity of more than 80% to the 16S rDNA sequence of Laurella R. No report is found on whether Kosakonia of the genus Kosakonia can cause patchouli bacterial wilt.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention mainly aims to provide a pathogenic strain causing patchouli bacterial wilt.

Another object of the present invention is to provide the use of the above pathogenic strains.

The invention carries out tissue soaking treatment on patchouli plants infected with bacterial wilt, and streaks the immersion liquid to a TTC flat plate. Selecting a bacterial strain with a morphology similar to that of a bacterial wilt pathogen colony, performing a pathogenicity tieback test on the patchouli to obtain a bacterial strain with strong pathogenicity, and identifying the bacterial colony morphology, a 16s rDNA gene and an rpoB gene to show that the bacterial strain belongs to the genus Kosakania (Kosakonia), is named Kosakonia sp.Pa82 and is a new pathogenic bacterial strain of the patchouli bacterial wilt. The pathogenic strain has strong pathogenicity on the patchouli, and has important significance on screening of disease-resistant germplasm of the patchouli, breeding of disease-resistant varieties and research and development of plant vaccines for preventing and treating bacterial wilt.

The purpose of the invention is realized by the following technical scheme:

the invention provides a pathogenic strain causing patchouli bacterial wilt, which is named as Kosakonia sp.Pa82 and is obtained by separating and purifying patchouli plants with typical bacterial wilt symptoms.

Deposit information of said strain Kosakonia sp.pa82: 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.

The strain Kosakonia sp.Pa82 is inoculated to a TTC plate by a three-line method and cultured at 30 ℃ for 24 hours, the strain has obvious fluidity on the TTC plate, and colonies are in a red round and plump shape and have white colonies, as shown in figure 1. After gram-staining, the cells were red, gram-negative, and short rods were formed, as shown in FIG. 2.

The 16s rDNA gene sequence of the strain Kosakonia sp.Pa82 is shown as SEQ ID NO: 1, the sequence length is 1538 bp. The rpoB gene sequence of the strain Kosakonia sp.Pa82 is shown as SEQ ID NO: 4, the sequence length is 4029 bp. The strain was identified as Kosaknia (Kosakonia) based on its morphological characteristics, 16S rDNA gene sequence and BLAST results of rpoB gene sequence in GenBank.

The strain Kosakonia sp.Pa82 is inoculated to healthy patchouli plants for pathogenicity test, and the result is shown in figure 3, the strain has strong pathogenicity to the patchouli and is a new pathogenic strain of patchouli bacterial wilt.

The strain Kosakonia sp.Pa82 is applied to researching the pathogenic mechanism of ralstonia solanacearum and excavating related genes of bacterial wilt virulence.

The strain Kosakonia sp.Pa82 is applied to screening of patchouli disease-resistant germplasm.

The strain Kosakonia sp.Pa82 is applied to breeding of a disease-resistant variety of pogostemon cablin.

The strain Kosakonia sp.Pa82 is applied to the development of plant vaccines for preventing and treating patchouli bacterial wilt.

The invention takes a strong pathogenic strain Kosakonia sp.Pa82 of patchouli bacterial wilt as a material, performs Tn5 transposon insertional mutation on the material by an electric shock transformation method, and obtains a mutant strain Pa82-87-1 with weakened pathogenicity by screening patchouli plants through a pathogenicity test. The Tn5 transposon insertion site gene of the low virulence mutant Pa82-87-1 is cloned, and the bioinformatics analysis result shows that the encoded protein of the transposon insertion site gene of the mutant 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 control effect of the mutant strain on the patchouli bacterial wilt can reach 71.70 percent.

Compared with the prior art, the invention has the following advantages and effects:

(1) the patchouli bacterial wilt new pathogenic strain Kosakonia sp.Pa82 provided by the invention provides a research material for researching bacterial wilt virulence related genes and pathogenic mechanisms and provides a theoretical basis for the control research of bacterial wilt diseases.

(2) Pa82 has strong pathogenicity on the pogostemon cablin, pathogenic strains with strong pathogenicity are used as stress pressure to screen resistant plants, a resistance mechanism of the plants is researched, a theoretical basis can be provided for cultivation of pogostemon cablin resistant varieties, and a research idea is provided for prevention and treatment research of other plant bacterial wilt.

(3) The invention takes a strong pathogenic bacterial strain Kosakonia sp.Pa82 of patchouli bacterial wilt as a material, adopts Tn5 transposon insertion mutation technology to construct a mutant strain of patchouli bacterial wilt Pa82, and obtains the mutant strain Pa82-87-1 with weakened pathogenicity by screening the pathogenicity of patchouli. The mutant strain is obtained by mutating pathogenic bacteria, the growth environment and the nutritional requirements of the mutant strain are the same as those of the pathogenic bacteria, and the function of plant vaccine is exerted by competing and inducing the potential disease resistance of plants. Biological control experiments of the low-pathogenicity mutant strain Pa82-87-1 on patchouli bacterial wilt show that the control effect can reach 71.70%. The new pathogenic strain Kosakonia sp.Pa82 for patchouli bacterial wilt, provided by the invention, has fast pathogenicity and strong pathogenicity to patchouli and high affinity to patchouli, and provides a valuable strain material for developing plant vaccines for preventing and treating patchouli bacterial wilt.

Drawings

FIG. 1 is the colony morphology of strain Pa82 on TTC plates.

FIG. 2 shows the cell morphology (1000X) of strain Pa82 under an oil lens.

FIG. 3 is the pathogenicity of the strain Pa82 on Pogostemon cablin plants.

FIG. 4 is a PCR electrophoretogram of 16s rDNA (A) and rpoB gene (B) of strain Pa 82; wherein M is DNA Marker of 5000 bp.

FIG. 5 is a phylogenetic analysis of the 16s rDNA gene of strain Pa82 and related strains.

FIG. 6 is a phylogenetic analysis of rpoB genes of strain Pa82 and related strains.

FIG. 7 Kan of resistant clones^rPCR 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. 8 shows the pathogenicity of the mutant strain Pa82-87-1 to Pogostemon cablin plant.

FIG. 9 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. 10 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. 11 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 double digestion result of Nhe I and Spe I.

FIG. 12 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, commercially available reagents and materials.

Example 1 isolation and purification of Agastache rugosa Ralstonia and determination of pathogenicity

(1) Separation and purification: collecting herba Agastaches plants with typical bacterial wilt symptom from field, soaking the diseased plants, streaking the extractive solution on TTC plate, culturing at 30 deg.C for 48 hr, and selecting single colony with colony morphology similar to that of Ralstonia solanacearum for purification and conservation.

Wherein, the culture medium of the TTC plate is acid hydrolyzed casein 1.0g/L, glucose 5.0g/L, peptone 10.0g/L, agar powder 10g/L and TTC 0.05g/L, and the pH value is 7.2.

(2) And (3) observing colony morphology: the purified and preserved strain was inoculated to TTC plates by the three-line method, cultured at 30 ℃ for 24 hours, and the bacterial colony morphology was observed. The colony is a smooth circular bulge, is red in the center, has a white colony ring, and has obvious fluidity as shown in figure 1.

(3) Pathogenicity determination: performing liquid culture on the purified and preserved strain, wherein the culture medium is Nutrient Agar (NA) liquid culture medium, performing shaking culture at 28 deg.C and 200r/min for 24 hr, diluting with sterile water by 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. Transferring the soaked plants to a greenhouse, and maintaining the humidity at about 85% and the temperature at 2%And observing and recording the disease condition of the plants for 1-7 days at the temperature of 8-30 ℃. The experiment was repeated 3 times. 1 strain with strong pathogenicity was obtained and recorded as Pa 82. The pathogenicity result of the Pa82 is shown in figure 3, the disease index of a plant treated by sterile water is 0, the disease speed of the plant inoculated with the strain Pa82 is high, and the disease index reaches 63.54% at the 2 nd day of inoculation; at inoculation 7d, the disease index reached 73.96%. The strain Pa82 has high affinity and strong pathogenicity to the host, and has the potential of being developed into a plant vaccine for preventing and treating the patchouli bacterial wilt.

Example 216 s rDNA sequence identification

Carrying out PCR amplification on a 16s rDNA gene by taking the genome DNA of the strain Pa82 as a template, wherein primers are as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3', 1541R: 5'-AAGGAGGTGATCCAGCCGCA-3' are provided. The PCR reaction used a 25. mu.L system: 27F/1541R (10. mu.M) 1. mu.L each, 1.5. mu.L of genomic DNA, ddH₂O9.0 mu L, Taq enzyme 12.5 mu L; the reaction parameters are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 100s, and 35 cycles; extension at 72 ℃ for 10 min. The PCR samples were subjected to agarose gel electrophoresis (FIG. 4A) and the samples were sequenced. The sequence results of the 16s rDNA gene were compared at NCBI, and strains with higher similarity and other plant pathogenic bacterial strains were selected, and a phylogenetic tree of 16s rDNA was constructed by the Neighbor-join method in MEGA5.1 software, as shown in FIG. 5. The results show that: the strain Pa82 is polymerized into one branch with the strain Kosakonia sp.CCTCC M2018092(Genbank Accession No. CP034225.1) and the strain Kosakonia cowanii Esp _ Z (Genbank Accession No. CP022690.1); BLAST alignment results show that the 16s rDNA gene sequence of strain Pa82 has 99.93% and 99.80% similarity to strain Kosakonia sp.cctcc M2018092 and strain Kosakonia cowanii Esp _ Z, respectively; pa82 was presumed to be a kosakholderia (Kosakonia) designated Kosakonia sp.pa 82.

The 16s rDNA gene sequence of the strain Pa82 is shown as SEQ ID NO: 1, the sequence length is 1538 bp.

Example 3 identification of rpoB Gene sequences

Carrying out PCR amplification on rpoB gene by taking the genome DNA of the strain Pa82 as a template, wherein primers are as follows: rpoB F: 5' -ATGGTTTACTCCTATACCGAGA-3', rpoB R: 5'-TTACTCGTCTTCCAGCTCGAT-3' is added. The PCR reaction used a 25. mu.L system: each of the rpoB F/R primers (10. mu.M) was 1. mu.L, genomic DNA was 1.5. mu.L, ddH₂O9.0 mu L, Taq enzyme 12.5 mu L; the reaction parameters are as follows: pre-denaturation at 94 ℃ for 2 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 100s, and 35 cycles; extension at 72 ℃ for 10 min. The PCR samples were subjected to agarose gel electrophoresis (FIG. 4B), and the samples were sequenced. The sequence results of rpoB gene were compared at NCBI, and strains with higher similarity and other plant pathogenic bacterial strains were selected, and a phylogenetic tree of rpoB gene was constructed by means of Neighbor-join in MEGA5.1 software, as shown in FIG. 6. The results show that: the strain Pa82 is closest to the strain Kosakonia sp.CCTCC M2018092(Genbank Accession No. CP034225.1) and the strain Kosakonia cowanii Esp _ Z (Genbank Accession No. CP022690.1); BLAST alignment results show that the rpoB gene sequence of the strain Pa82 has more than 99% similarity with the strain Kosakonia sp.cctcc M2018092 and the strain Kosakonia cowanii Esp _ Z. Pa82 was presumed to be a kosakholderia (Kosakonia) designated Kosakonia sp.pa 82.

The rpoB gene sequence of the strain Pa82 is shown in SEQ ID NO: 4, the sequence length is 4029 bp.

Therefore, Pa82 was identified as kosakholderia (Kosakonia) and named Kosakonia sp.pa82 based on the morphological characteristics of strain Pa82 and BLAST results of the 16S rDNA gene sequence and rpoB gene sequence in GenBank.

Deposit information of said strain Kosakonia sp.pa82: 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 4

Construction of transposon insertion mutant of strain Kosakonia sp.Pa82 Tn5

Transposon kit EZ-Tn5^TM＜KAN-2＞Tnp Transposome^TMKit, purchased from Epicentre, USA, is Tn5 transposon (transposon) and transposase (transposase) complex, 1221bp in total length, carrying a transposon in the middleKanamycin resistance (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 Kan^rPCR amplification of the gene (P1: 5'-GGTGCGACAATCTATCGA-3' and P2: 5'-CTCATCGAGCATCAAATG-3') and electrophoretic detection results are shown in FIG. 7, and Tn5 transposon (positive control) and resistant clone showed specific bands at about 700bp, while the wild strain did not amplified the corresponding bands, indicating that Tn5 transposon insertion mutant of strain Kosakonia sp.Pa82 was obtained by shock transformation.

(II) screening of Low-pathogenicity mutant Strain of Kosakonia sp.Pa82

And (3) carrying out pathogenicity screening by taking transposon insertion mutant strains of the series of strains Pa82 obtained in the step (I) as test strains and taking a 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 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 attack conditions of the plants for 1-7 d. 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 mutant strain Pa82-87-1 to the cablin potchouli herb is shown in figure 8. Disease of plant treated with sterile waterThe disease index is 0, the disease index of a plant inoculated with the wild strain Pa82 is the highest and reaches more than 80% at the 7 th day of inoculation, and the disease index of a plant inoculated with the mutant strain Pa82-87-1 is obviously reduced compared with that of the wild strain. Through pathogenicity screening, a low-pathogenicity mutant strain Pa82-87-1 is obtained.

(III) cloning of Low-virulence mutant Pa82-87-1 transposon insertion site Gene

(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. 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 result is shown in FIG. 9, wherein 1 band was amplified from the low virulence mutant strain Pa82-87-1, and the target band was not amplified from the negative control strain Pa 82. And performing sequence determination on 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 flanking sequences of a Tn5 transposon insertion site by BLAST: 87-1-F: 5' -CTAGCTAGCATGTTGAACAGAATTACGGTCAAG-3' (underlined Nhe I cleavage site), 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 ℃ 10s, annealing at 58 ℃ for 15s, extending at 72 ℃ for 1min, and performing 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 10, 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) performing gel cutting recovery on the PCR amplification product of the wild strain 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 11, 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: 11, 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 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: year 2020, 7, 16.

(IV) Low-pathogenicity mutant strain Pa82-87-1 control effect test on 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. Irrigation of biocontrol group to patchouli plantsPre-inoculating low-pathogenicity mutant Pa82-87-1, and after 3d, injuring roots and soaking to inoculate wild strain Pa 82. 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. 12, after 7d of inoculation, 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

<110> Guangzhou college of traditional Chinese medicine (Guangzhou institute of traditional Chinese medicine)

<120> pathogenic strain causing patchouli bacterial wilt and application thereof

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gcaagcgtta atcggaatta ctgggcgtaa agcgcacgca ggcggtctgt caagtcggat 600

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gaggggggta gaattccagg tgtagcggtg aaatgcgtag agatctggag gaataccggt 720

ggcgaaggcg gccccctgga cgaagactga cgctcaggtg cgaaagcgtg gggagcaaac 780

aggattagat accctggtag tccacgccgt aaacgatgtc gacttggagg ttgtgccctt 840

gaggcgtggc ttccggagct aacgcgttaa gtcgaccgcc tggggagtac ggccgcaagg 900

ttaaaactca aatgaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg 960

atgcaacgcg aagaacctta cctggtcttg acatccacag aactttccag agatggattg 1020

gtgccttcgg gaactgtgag acaggtgctg catggctgtc gtcagctcgt gttgtgaaat 1080

gttgggttaa gtcccgcaac gagcgcaacc cttatccttt gttgccagcg gtccggccgg 1140

gaactcaaag gagactgcca gtgataaact ggaggaaggt ggggatgacg tcaagtcatc 1200

atggccctta cgaccagggc tacacacgtg ctacaatggc gcatacaaag agaagcaatc 1260

tcgcgagagc tagcggacct cataaagtgc gtcgtagtcc ggattggagt ctgcaactcg 1320

actccatgaa gtcggaatcg ctagtaatcg tgaatcagaa tgtcacggtg aatacgttcc 1380

cgggccttgt acacaccgcc cgtcacacca tgggagtggg ttgcaaaaga agtaggtagc 1440

ttaaccttcg ggagggcgct taccactttg tgattcatga ctggggtgaa gtcgtaacaa 1500

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atggtttact cctataccga gaaaaaacgt attcgtaagg attttggtaa acgtccacaa 60

gtactggatg ttccctatct cctttctatc cagcttgact cgttccagaa gttcatcgag 120

caagatcctg agggccagta cgggctcgaa gcggcattcc gctccgtgtt cccgatcaag 180

agctacagcg gcaattcgga actgcaatac gtcagctacc gtcttggcga acccgtattt 240

gacgttaaag agtgtcaaat ccgtggtgtg acgtactccg ccccgctgcg cgtaaaactg 300

cgtctggtga tctacgagcg cgaagcgccg gaaggcaccg taaaagacat taaagaacaa 360

gaagtctaca tgggcgaaat tccgctcatg accgacaacg gtacctttgt tatcaacggt 420

actgagcgtg ttatcgtttc tcagctgcac cgtagcccgg gcgtcttctt tgacagcgac 480

aagggtaaaa cccactcttc cggtaaggtg ctctataacg cacgtatcat tccttaccgt 540

gggtcatggc tggactttga gttcgatccg aaagacaacc tgttcgttcg tatcgaccgt 600

cgtcgtaagc tgccggccac catcattctg cgcgcgctga actacaccac tgaacagatc 660

cttgacctgt tctttgaaaa agtggtcttt gagatccgcg acaacaagct gcagatggaa 720

ctggtaccgg aacgtctgcg tggcgaaacc gcctccttcg atatcgaagc caacggcaaa 780

atgtatgttg agaaaggccg ccgcatcacc gcgcgccaca ttcgtcagct ggaaaaagac 840

gaaatccaac atatcgaagt gccggttgag tacatcgcag gcaaagttgc ggcgaaagac 900

tacgttgacg aatccactgg cgagctgatc tgcccggcca acatggagct gtcgctcgat 960

ctgctggcta aactgagcca ggctggtcac aaacgtatcg aaacgctgtt caccaacgat 1020

ctggatcacg gcccgtacat ctctgaaacc gtacgcgtcg acccaactaa cgatcgtctg 1080

agcgcgctgg tagaaatcta ccgcatgatg cgcccgggtg agccgccgac tcgcgaagcg 1140

gctgaaagcc tcttcgaaaa cctgttcttc tccgaagacc gctacgacct ttctgcggta 1200

ggccgcatga agttcaaccg ttctctgctg cgcgacagca ttgaaggttc cggtatcctg 1260

agcaacgacg acatcatcga agtgatgaag aagctcatcg gtatccgtaa cggcatcggt 1320

gaagtggatg atatcgacca cctcggcaac cgtcgtatcc gttccgtagg cgaaatggcg 1380

gaaaaccaat tccgcgttgg cctggtgcgt gtagagcgtg cggtgaaaga gcgtctctct 1440

ctgggcgatc tggataccct aatgccgcag gacatgatca acgccaagcc gatttcggcg 1500

gcggtgaaag agttcttcgg ctccagccag ctctcccagt ttatggatca gaacaacccg 1560

ctgtctgaga ttacgcacaa acgtcgtatc tctgcacttg gcccgggcgg tctgacccgt 1620

gagcgcgcag gctttgaagt tcgagacgta cacccgaccc actacggtcg cgtatgtcca 1680

atcgaaacgc ctgaaggtcc gaacatcggt ctgatcaact ccctgtccgt ctatgcacag 1740

actaacgaat atggcttcct cgaaacgccg taccgtaaag tgactgacgg cgtggtgact 1800

gacgagatcc attacctctc tgcaattgaa gagggtaact acgttatcgc tcaggcgaac 1860

accaacctga cggaagaagg gcgttttgca gaagatctgg taacctgccg tagcaaaggc 1920

gaatcaagcc tcttcagcgc agaccaggtt gactacatgg acgtatccac ccagcaggtg 1980

gtttccgtcg gtgcatccct gattccgttc ctggaacacg atgacgccaa ccgtgcattg 2040

atgggtgcga acatgcaacg tcaggcggtt ccgactctgc gcgctgataa gccgctggtt 2100

ggtaccggta tggaacgtgc tgttgccgtt gactccggcg taactgccgt tgcgaaacgt 2160

ggcggtaccg ttcagtacgt ggatgcttcc cgtatcgtta tcaaagttaa cgaagacgag 2220

atgtacccgg gcgaagcagg tatcgacatc tataacctga ccaaatacac ccgttctaac 2280

cagaacacct gcatcaacca gatgccgtgt gtttacctgg gtgagccgat tgagcgcggc 2340

gacgtgctgg cagacggtcc gtccaccgac cttggtgaac tggcgctcgg tcagaacatg 2400

cgcgtagcgt tcatgccgtg gaacggttac aacttcgaag actccatcct cgtctccgag 2460

cgtgtggttc aggaagatcg tttcaccact attcacatcc aggaactggc ttgtgtgtcc 2520

cgtgacacca aactggggcc ggaagagatc accgctgaca tcccgaacgt gggtgaagct 2580

gcgctctcca aactggatga atccggtatc gtttacatcg gtgcagaagt gaccggcggc 2640

gacattctgg ttggtaaggt aacgccgaaa ggtgaaaccc agctgacgcc agaagagaaa 2700

ctgctgcgcg cgatcttcgg tgagaaagcg tctgacgtta aagactcttc tctgcgcgta 2760

ccaaacggtg tttccggtac ggttatcgac gttcaggtct tcacccgcga tggcgtggaa 2820

aaagacaaac gtgcgctgga aatcgaagaa atgcagctca aacaggcgaa gaaagacctg 2880

tctgaagaac tgcagatcct cgaagctggc ctgttcagcc gtatccactc cgtgctggta 2940

tccggtggcg ttgaagctga gaagctcgac aaactgccgc gcgaccgctg gctggaactc 3000

ggcctgaccg acgaagcgaa acagaatcag ctggaacagc tggctgagca gtacgacgaa 3060

ctgaaacacg agttcgagaa aaagctcgaa gcgaaacgcc gtaagatcac tcagggcgac 3120

gatctggcac cgggcgtgct gaagattgtt aaggtttatc tggccgttaa acgtcagatc 3180

cagcctggtg ataagatggc aggtcgtcac ggtaacaagg gtgttatctc caagatcaac 3240

ccgatcgaag atatgccata cgatgaaaac ggcacgccgg tagacatcgt actgaacccg 3300

ctgggcgtac cgtctcgtat gaacatcggt cagatccttg aaacccacct gggtatggct 3360

gcgaaaggta ttggcgacaa gattaacgcc atgctgaaac agcagcagga agtcgcgaaa 3420

ctgcgcgagt tcatccagcg tgcatacgat ctgggcgctg acgttcgtca gaaagtcgac 3480

ctgagcacct tcagcgatga cgaagtgctg cgtctggctg aaaacctgcg taaaggcatg 3540

ccgatcgcaa cgccggtctt cgacggtgcg aaagagtctg aaatcaagga actgttacag 3600

ctgggtggcc tgccgacttc cggtcagatc acactgttcg acggtcgtac cggtgagcag 3660

ttcgagcgcc aggttaccgt tggctacatg tacatgctga aactgaacca cctggttgat 3720

gacaaaatgc atgcgcgttc caccggttcc tacagcctgg ttactcagca gccgctgggt 3780

ggtaaggcgc agttcggtgg tcagcgcttc ggggagatgg aagtgtgggc gctggaagca 3840

tatggcgcgg catacaccct gcaggaaatg ctcaccgtta agtctgatga cgtgaatggt 3900

cgtaccaaga tgtataagaa catcgtggac ggcaaccatc agatggaacc ggggatgcca 3960

gagtccttca acgtactgtt gaaagagatt cgttcgctgg gtatcaacat cgagctggaa 4020

gacgagtaa 4029

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> rpoB F

<400> 5

atggtttact cctataccga ga 22

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> rpoB R

<400> 6

ttactcgtct tccagctcga t 21

<210> 7

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P1

<400> 7

ggtgcgacaa tctatcga 18

<210> 10

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> P2

<400> 10

ctcatcgagc atcaaatg 18

<210> 8

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> KAN-2 FP-1

<400> 8

acctacaaca aagctctcat caacc 25

<210> 9

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> KAN-2 RP-1

<400> 9

gcaatgtaac atcagagatt ttgag 25

<210> 11

<211> 2613

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Low virulence mutant Pa82-87-1 transposon insertion site Gene nucleotide sequence

<400> 11

atgttgaata gaattacggt caagctgccg gtagacggcc tgcttttctg gaaactcacc 60

ggtcgcgagg cgctgtctga agcgtttgct ttttccctga cggtgctggg caccgacgcg 120

cgtatcgacc gcagccagct tctcggccag ccggtgacgg tgaatgtgcc gacgcagggg 180

attgccggcg gcacgcgcca cttcaacggt aaaatcaccc gcgtggcggt gagcgccacg 240

gagatgtccg ggacgcggta tgcggtctac cggctgacgg tggagccgga cgtgtggccg 300

atgaagcgcg accgcaacct gcgcatcttc caggagcaga cggtgccgca gattgtgaag 360

accctgcttg gggaatacca ggtgaacctt gaggaccgcc tgaccggcag ctaccggagc 420

tggggctact gcgtgcagta ccaggagagc agttttgact tcatcagccg tctgatggag 480

cttgagggca ttgcctacca cttcagtcac gaagccgacc ggcacgtgat ggtgctgacg 540

gacgccgcca ccgaacacca gcccttcagc gggtacgaaa ccattcccta ccacccgacg 600

ccgtccggcg gcagcaccag cgaagagggc atcagccagt gggcgctgga ggacagcgtg 660

acaccgggca tctacagcct tgacgactac gacttccgca aaccgaacgc atggctgttc 720

caggcccggc agaatccggc ctcgccgcag ccggggagca ttgacgtcta tgactggccg 780

ggccgctttg tcgagcacgg tcacggtgaa tattatgccc gcatccgcca ggagcgctgg 840

caggtggagc accagcagat tcaggccacc gccacggcag cgggtattgc gccgggtcac 900

acctttgcgc tctacaacgc gccgttcttc agcgataacg gcgagtacct gaccaccgag 960

gcgaactact tcctcgagga gaaccgctac gccagcgggt cagacggcga gacggtgcac 1020

cgcaccgatt tcaccgtcat tccctcgtcg gtggtgttcc gcccggcgcc ggtaacagcg 1080

tggccgaaaa cccacggccc gcagacggca aaagtggtcg gcccgcaggg cgagagtatc 1140

tggacggaca aatatggccg gataaaggtg aagttccact gggaccgtca ggcgaaaggg 1200

gatgacacca gctcctgctg ggtgcgcgtg tcgagcgcgt gggcaggcca gggcttcggc 1260

ggggtacaaa tcccgcgcgt gggggatgag gtggtgattg actttatcaa cggcgacccg 1320

gaccgtccga tcgtgacagg gcgcgtctac aacgaagcga gcatgccgcc atggtcactg 1380

cccgctgccg caacacagat gggctttctc agccgctcaa aagatggctc ggttgataac 1440

gccaacgccc tacgctttga agataaagcg ggggaggagc aggtgtggat tcaggccgag 1500

cgtaatctgg atgtacatgt aaaaaacgat gcttcccatt ccattggcag taatcacagt 1560

cattatgtgc gcaaaaacgc gcttcatcgc gttgaagcca accagaccca tgtcgtaaag 1620

gggcaaacag aaaacctcac cggaaaaggc aagcttgatg ctgcggttga gcaatttatt 1680

atcgcctctg gcacgcagct tcggctggtc tccggtgata gtgcgattga gttgaacgct 1740

aacggcaaaa tcaatctgat cggtaaatcg tttaactttt ttgttgaaga ggatgggcat 1800

ataaccaccg gtggtaaatt gcatctcaac actgaaggaa cacctgcgcc aaccgcagca 1860

ccgggatcgg gtcataaagg cgatattcac acagccgtag agcgattttt ctctccgcaa 1920

agtgaggtaa aacatgccgc gcctgtggca actgccactg cgtctcctcc aactcaaaaa 1980

ccgatcgcga aatataaagc accgccgccg ctgaaagggg attatgttta cagtaatgaa 2040

aaaagcaaga aactctttat gccttttagc gatggtgtga tagagaaaat aaacagctca 2100

cctaaaatgc aacaagattt gaaaaaattg ctggatgatg agtggaatat agcaccaaac 2160

gtgccaggtt caggctcctg gacagatact gataacaaac ttatggtgct ggatccagaa 2220

catatggcga atgacgatga ggctgttatt acccttgccc acgaattggg gcacgccact 2280

agcccttata ctcatgattt aacatctcgg accaattttg tgaatagcat gctcaatgat 2340

gagggtcatg ccacgttgaa tgaaattgag gttaggcgag aaatttatca caatatggga 2400

gtagatatag gctcagtctt tggatcacaa aacgagagta agtatattaa aatatttaaa 2460

gatatggata gcggtaagat tacacgtaac gcagcaagca aagcaatcgg tgaaatatat 2520

agacggggag aagttgcttc cagttcgctt acagacgagg tttatgagga acattatggc 2580

aagatgtatg atgctaataa gtcgaaaccg taa 2613

<210> 12

<211> 33

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 87-1-F

<400> 12

ctagctagca tgttgaacag aattacggtc aag 33

<210> 13

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> 87-1-R

<400> 13

ggactagttt acggtttcga ctttttagca tc 32

Claims (4)

1. A pathogenic strain causing patchouli bacterial wilt is characterized in that: the name of the bacterium Coxsackii (Kosakoniasp.) Pa82, deposited at 16.7.2020 in the Guangdong province collection of microorganisms of Guangdong province, No. 59 building, No. 5 building, Guangdong province institute of microorganisms of Michelia, Guangzhou, with the collection number: GDMCC No: 61047.

2. the pathogenic strain of claim 1, for use in screening for disease-resistant germplasm of Pogostemon cablin.

3. The pathogenic strain of claim 1, for use in breeding a disease-resistant variety of Pogostemon cablin.

4. The application of the pathogenic bacteria strain of claim 1 in preparing plant vaccines for preventing and treating patchouli bacterial wilt.

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