CN113913338A - Separation and identification method of pathogenic bacteria of bacterial black spot of tobacco - Google Patents
Separation and identification method of pathogenic bacteria of bacterial black spot of tobacco Download PDFInfo
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Abstract
The invention discloses a method for separating and identifying pathogenic bacteria of bacterial black spot of tobacco, which comprises the following steps: s1: collecting disease samples; s2: separating pathogenic bacteria; s3: culturing and screening pathogenic bacteria; s4: preparing bacterial colony PCR bacterial suspension; s5: PCR amplification and identification; s6: and (3) amplifying and verifying the 16S rRNA gene. The method can quickly and accurately obtain the pathogenic bacteria of the black spot of the tobacco, effectively eliminate other mixed bacteria, improve the separation and purification efficiency and ensure that the separation and purification rate is more than 87%; the specific primers of the pathogenic bacteria of the bacterial black spot are adopted to carry out PCR amplification confirmation on the gray bacterial colonies, so that the selectivity of the target bacterial strains is enhanced, and the pathogenic bacteria can be rapidly identified.
Description
Technical Field
The invention relates to the technical field of tobacco disease identification, in particular to a method for separating and identifying pathogenic bacteria of bacterial black spot of tobacco.
Background
The tobacco leaves are the main positions for harvesting the tobacco, the quality of the cigarette products is directly related to the quality of the tobacco leaves, the importance of the quality is self-evident, and meanwhile, the tobacco leaves are the parts which are most seriously affected by diseases and insect pests. Tobacco diseases are mainly divided into three types of diseases, namely virus, fungus and bacteria. Wherein, the leaf diseases caused by pathogenic bacteria comprise: bacterial leaf spot in seedling stage caused by Pseudomonas amygdalinensis (wildfire of tobacco), angular leaf spot (angular leaf spot of tobacco), Pseudomonas aeruginosa (P. aeruginosa), bacterial leaf blight caused by Pectinopus carotovorus subsp. brasiliensis (Pectinopus carotovorus subsp. brasiliensis), bacterial leaf spot caused by Xanthomonas campestris (Xanthomonas campestris) infection, and leaf sisal (decending of tobaco) caused by Bacillus cereus.
Among them, wildfire and angular leaf spot are common bacterial diseases of tobacco. The tobacco wildfire disease mainly occurs in the field, the symptom is initially water stain-shaped small spots, the centers of the spots become brown with deepening of the disease and enlargement of the spots, the spots are threaded, obvious yellow-green halos are formed around the spots, the wildfire disease can be spread in the field through wind and rain and invaded from natural orifices or wounds, and the wildfire disease has the characteristics of strong outbreak and large destructiveness. Angular leaf spot is mostly generated in the middle and later growth stages, leaves are polygonal or irregular, the edges are clear, so the angular leaf spot is called, as the disease deepens, the disease spots are enlarged, the disease spots are mutually combined, the disease spots crack and fall off after being dried, the leaves are crushed, the bacterial pus overflows on the back of the leaves, and a film is formed after the leaves are dried.
In 9-month middle ten-day of 2019, the inventor of the application finds a new tobacco leaf disease on Yunyan 105 and Yunyan 87 planted in great east and rural areas of Li Jiang city, Yunnan province, the bacterial black spot of tobacco is a new domestic disease found in Yunnan province in recent years, the disease damages the whole tobacco leaf, and the lower leaves are heavy; most of the disease spots are irregular black spots, the black spots are large and sometimes limited by veins and collateral vessels, the surfaces of the disease spots have no mildew layers and are few in halos, the disease spots are obviously different from known angular leaf spot and wildfire, and the disease spots are called as tobacco bacterial black spot diseases.
In view of the above, there is a need to develop a method for isolating and identifying pathogenic bacteria of bacterial black spot of tobacco to solve the above-mentioned problems.
Disclosure of Invention
The invention aims to provide a method for separating and identifying pathogenic bacteria of bacterial black spot of tobacco, which can quickly and accurately obtain the pathogenic bacteria of the bacterial black spot of tobacco, effectively eliminate other mixed bacteria and improve the separation and purification efficiency; the specific primer of the bacterial black spot pathogenic bacteria of the invention confirms gray bacterial colonies, and can rapidly identify the pathogenic bacteria.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for separating and identifying pathogenic bacteria of bacterial black spot of tobacco, which comprises the following steps:
s1: collecting disease samples: collecting diseased leaves with tobacco bacterial black spot disease spots in the field, putting the diseased leaves into a sampling bag, and bringing the sampling bag back to a laboratory;
s2: and (3) pathogenic bacteria separation: washing the tobacco bacterial black spot disease leaves with disease spots in the step S1 for 3-5 times by using flowing tap water, taking a plurality of fresh disease spots at disease-health junctions, and rinsing after disinfecting in a sterile culture dish; cutting the rinsed lesion spots into a plurality of tissue blocks, placing the tissue blocks in a sterilized culture dish containing 10-20mL of sterile water, soaking for 5-10min, and shaking the culture dish to uniformly mix bacterial liquid after milky bacterial liquid overflows around the tissue blocks to obtain bacterial suspension;
s3: and (3) pathogenic bacteria culture and screening: sucking 30-50 mu L of the bacterial suspension obtained in the step S2 into an NA culture medium plate by using a pipettor, streaking and diluting the bacterial suspension by using a sterile toothpick, and drying the diluted bacterial suspension by placing the diluted bacterial suspension on an ultra-clean workbench; drying, placing in an incubator at 28-30 ℃ for dark culture for 36-49h, culturing for 23-25h to form a plurality of small single colonies, culturing for 47-49h to obtain light gray single colonies, and placing the light gray single colonies in an environment at normal temperature or 4 ℃ for 2-5d to obtain dark gray single colonies;
s4: preparing bacterial colony PCR bacterial suspension: adding 10-100 mu L of sterile water into a sterilized centrifuge tube, picking a dark gray single bacterial colony in the step S3 by using a sterilized toothpick, placing the bacterial colony in a vortex oscillator, and vibrating and uniformly mixing to prepare bacterial colony PCR bacterial suspension;
s5: PCR amplification and identification: taking 0.5-2 mu L of bacterial colony PCR bacterial suspension in the step S4, carrying out bacterial colony PCR amplification by using tobacco bacterial alternaria alternata specific primers PcF and PcR, and obtaining a PCR product after the PCR amplification is finished; taking 5-10 mu LPCR product, loading the product into a gel hole of 0.8-1.5% agarose gel, separating for 30-45min under the constant pressure of 120-145V, and staining for 10-20min in EB nucleic acid staining solution containing 5-10 mu g/mL; placing the mixture on a gel imager to obtain the separation condition of a PCR product, wherein if the mixture is the tobacco bacterial macula nigra, a specific amplification strip exists between 750 bp and 1000 bp;
s6: 16S rRNA gene amplification and validation: and (3) shaking the bacteria with the positive colony PCR amplification by using an LB culture medium for 24h, extracting genome DNA by using a bacterial genome DNA extraction kit, carrying out PCR amplification by using 16S rRNA gene amplification universal primers 27F and 1492R, sequencing the amplified product, and analyzing the sequencing result, wherein the amplified band size is about 1540 bp.
By adopting the method, various types of bacterial colonies are often formed on the surface of a culture medium due to the influence of saprophytic bacteria during the separation of pathogenic bacteria, and the pathogenic bacteria are difficult to quickly identify; according to the characteristic of light grey to dark grey presented by the colony morphology of the pathogenic bacteria, the identification degree of the pathogenic bacteria is improved. The pathogenic bacteria of the black spot are rapidly separated and identified, so that the field management and symptomatic medicine administration are facilitated.
Preferably, the diseased leaf in the step S1 is an irregular large black spot, the surface of the black spot has no mildew layer, the halo at the edge of the black spot is not obvious, and the edge of the black spot is limited by the veins or the collateral veins.
By adopting the method, the symptoms of the tobacco bacterial black spot are obviously different from the common wildfire disease and angular leaf spot, the disease spot of the tobacco bacterial black spot is black and large and is irregular, but is often limited by veins and collateral veins to be in a patch shape, the surface of the disease spot has no mildew layer, and the halo is not obvious.
Preferably, more than 10 fresh lesions are taken in the step S2; the disinfection is to disinfect the surface of the steel tube by using 75% alcohol for 5s and then disinfect the steel tube by using 0.5% sodium hypochlorite for 5 min; the rinsing is 3 times of rinsing with sterile water; the tissue block has a size of 0.5 × 0.5cm2。
Preferably, the method for preparing the NA medium plate in step S3 includes: respectively weighing 18g of nutrient broth and 15.0g of agar, adding distilled water to a constant volume of 1L, autoclaving at 121 ℃ for 20min, placing in a 55 ℃ water bath kettle, adding 23-28mL of culture medium into each sterile culture dish when the temperature of the culture medium is reduced to about 60 ℃, and condensing to obtain an NA plate.
Preferably, the sequence of the primer PcF in the step S5 is 5'-CCGTTCATCGTCATCGACCT-3', and the sequence of the primer PcR is 5'-CTGTCCCACATGATCTGGGT-3'.
By adopting the method, the provided specific primer for the pathogenic bacteria of the bacterial black spot of the tobacco provides a feasible scheme for the rapid and accurate identification of the pathogenic bacteria; the grey colonies are confirmed by adopting the specific primers of the pseudomonas chicory, so that pathogenic bacteria can be quickly identified.
Preferably, the reaction system identified by PCR in step S5 is 25 μ L, and the reaction system consists of 12 μ L2 XEs TaqMasterMix (Dye), 12 μ L ddH2O, 0.5 muL of 10 muM PcF, 0.5 muL of 10PcR and 0.5-2.0 muL of bacterial suspension; the colony PCR reaction condition is pre-denaturation at 94 ℃ for 2min, the amplification cycle comprises denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, the amplification cycle comprises 30 cycles, and finally extension at 72 ℃ for 2min, and the PCR product is stored at 16 ℃ or directly used for agarose gel electrophoresis.
Preferably, there is a specific amplification band between 750 and 1000bp described in step S5, specifically a band located at 897 bp.
Preferably, the PCR reaction system for the 16S rRNA gene in the step S6 is 16. mu.L ddH2O, 3 mu.L dNTP MIX, 5 mu. L Q5 Reaction Buffer, 0.3 mu. L Q5 DNApolymerase, 0.5 mu.L 27F, 0.5 mu.L 1492R and 0.5-2 mu.L genomic DNA, wherein the PCR amplification program is pre-denaturation at 98 ℃ for 30s, the amplification cycle comprises denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 45s, amplification is carried out for 30 cycles, and finally extension at 72 ℃ is carried out for 2 min.
Preferably, the pathogenic bacterium determined in step S6 is Pseudomonas chicory (Pseudomonas cichorii). The diseased spots on the tobacco leaves are black and large, the diseased spots are irregular, but are often limited by veins and collateral veins to be in a plaque shape, the surfaces of the diseased spots have no mildew layer, and the tobacco bacterial black spot disease (bacterial black spot obacaco) caused by the infection of pseudomonas cichorii (P.cichororii) can be judged if the halo is not obvious.
In summary, compared with the prior art, the invention has the advantages that:
1. the invention utilizes the characteristic that the pathogenic bacteria of the tobacco bacterial black spot form light gray to dark gray bacterial colonies on a nutrient broth culture medium, and combines the specific primers of the pathogenic bacteria of the tobacco bacterial black spot, thereby enhancing the selectivity of target strains, having high purification speed, high efficiency and high separation and purification rate, and ensuring the pathogenic bacteria of the black spot.
2. According to the invention, the characteristic from light grey to dark grey is presented according to the colony morphology of pathogenic bacteria, so that the identification degree of the pathogenic bacteria is improved; during colony PCR identification, the initial judgment can be carried out according to the change of the colony color, other interference strains without the characteristic are eliminated, and the target selectivity is strong.
3. The specific primer of the pathogenic bacteria of the tobacco bacterial black spot provides a feasible scheme for the rapid and accurate identification of the pathogenic bacteria; the specific primers are adopted to confirm the gray colonies, so that pathogenic bacteria can be quickly identified.
4. The bacterial black spot caused by the infection of the pseudomonas chicory (P.cichororii) is identified by separation, culture, screening and identification.
5. The characteristics and specific symptoms of the bacterial black spot of the tobacco disclosed by the invention provide an important basis for accurately identifying diseases and successfully separating pathogenic bacteria; the leaves with typical disease symptoms are collected for separation, so that the separation probability of pathogenic bacteria can be improved.
6. The separation and identification method of the present invention is not only rapid, but also simple and easy to implement, and the method is very easy to be mastered by those skilled in the art.
Drawings
FIG. 1 is a flow chart of the present invention;
FIG. 2 is a typical symptom of bacterial black spot of tobacco of the present invention;
FIG. 3 shows that the center of the bacterial colony of the bacterial black spot pathogen of tobacco is gray after the bacterial colony is cultured for 48 hours on the NA culture medium;
FIG. 4 is a PCR assay of suspected pathogens of tobacco bacterial black spot in accordance with the present invention;
FIG. 5 is a 16S rDNA sequence phylogenetic tree of Nicotiana tabacum LJ2 constructed based on the proximity method.
Detailed Description
The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1
The black spot disease sample in the embodiment is 8 parts of tobacco bacterial black spot disease samples collected from Yunnan Lincang and Lijiang in 8-9 months in 2020.
1.1 Collection of disease specimens
Typical diseased leaves of the bacterial black spot of the tobacco are collected from the field, the selected diseased leaves have large irregular black spots, the surfaces of the diseased spots have no mildew layer, the edge halos are not obvious, the edges are sometimes limited by veins and side veins, the diseased leaves are generally easy to occur in high-altitude areas with damp and low temperature, and the typical symptoms of the bacterial black spot of the grass are shown in figure 2. The whole leaf was placed in a sampling bag and taken back to the laboratory.
1.2 isolation of pathogenic bacteria
Washing fresh leaf scab with flowing tap water for 3 times, taking off fresh scab at the juncture of the scab with sterile blade, placing 15 scabs in sterile petri dish, sterilizing with 75% alcohol surface for 5s, sterilizing with 0.5% sodium hypochlorite for 5min, rinsing with sterile water for 3 times, and cutting sterilized scab with sterile blade into pieces of 0.5 × 0.5cm2And (3) placing the tissue blocks in a sterilized culture dish containing 10mL of sterile water, soaking for 10min, and shaking and uniformly mixing the bacterial suspension after milky bacterial liquid overflows around the tissue blocks.
1.3 pathogen culture and selection
Sucking 30 mu L of bacterial suspension on an NA culture medium plate by using a pipette, marking and diluting the bacterial suspension by using a sterile toothpick, placing the diluted bacterial suspension on an ultra-clean workbench for drying, placing the diluted bacterial suspension in an incubator at 28 ℃ for dark culture for 48h, and finding that the culture for 48h is optimal by the inventor through a large number of experiments.
The experimental results are as follows: suspected pathogenic bacteria were separated from 8 samples of tobacco bacterial black spot collected at lincang, and the colonies appeared light gray at the initial stage, and dark gray after being placed in a refrigerator at room temperature or 4 ℃ for 2 days, as shown in fig. 3.
1.4 identification of pathogenic bacteria of bacterial black spot of tobacco
1.4.1 bacterial suspension preparation: and adding 100 mu L of sterile water into a sterilized centrifuge tube, picking single bacterial colonies with the bacterial colony centers from light gray to dark gray by using a sterilized toothpick, placing the single bacterial colonies in the sterile water, and vibrating and uniformly mixing the single bacterial colonies on a vortex oscillator to prepare bacterial colony PCR bacterial suspension.
1.4.2PCR amplification
Taking 1 mu L of colony PCR bacterial suspension, and carrying out colony PCR amplification by using a tobacco bacterial alternaria alternata specific primer PcF/PcR, wherein the sequence of the primer PcF is 5'-CCGTTCATCGTCATCGACCT-3', and the sequence of the primer PcR is 5'-CTGTCCCACATGATCTGGGT-3'. The reaction system for colony PCR identification is 25 μ L, and the reaction system consists of 12 μ L2 XEs TaqMasterMix (Dye), 12 μ L ddH2O, 0.5. mu.L of 10. mu.M PcF, 0.5. mu.L of 10PcR and 1. mu.L of bacterial suspension; the colony PCR reaction condition is pre-denaturation at 94 ℃ for 2min, the amplification cycle comprises denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, amplification for 30 cycles, and finally extension at 72 ℃ for 2 min.
After the PCR amplification was completed, 5. mu.L PCR product was applied to a 1% agarose gel well, separated at a constant pressure of 120V for 45min, and stained in EB nucleic acid staining solution containing 5. mu.g/mL for 10 min.
The experimental results are as follows: as a result, as shown in FIG. 4, 3 single colonies each having light gray to dark gray were selected from 8 samples collected at lincang and Lijiang and colony PCR amplification was performed. The result shows that specific amplified bands between 750 and 1000bp can be amplified from 21 single colonies, the specific size is 897bp, only 3 single colonies have no amplified band, and the positive detection rate is 87.50%. The results show that the screening method combining the colony color change of the pathogenic bacteria and the specific primers can specifically distinguish suspected pathogenic bacteria and improve the accuracy of pathogenic bacteria separation.
1.5 verification of tobacco bacterial black spot pathogen:
16S rRNA gene amplification: PCR amplification of coloniesThe positively amplified strain LJ2 was cultured on LB medium for 24 hours, and genomic DNA was extracted using a bacterial genomic DNA extraction kit (cat # D3107, Kyoho Biotech, Guangzhou) and PCR-amplified with the 16S rRNA gene-amplified universal primer 27F/1492R, the 27F primer sequence being 5'-AGAGTTTGATCCTGGCTCAG-3' and the 1492R primer sequence being 5'-CGGTTACCTTGTTACGACTT-3'. The reaction system for PCR amplification of the 16S rRNA gene is 25 mu L and consists of the following components: 16 μ L ddH2O, 3. mu.L dNTP MIX, 5. mu. L Q5 reaction buffer, 0.3. mu. L Q5 DNA Polymerase, 0.5. mu.L 27F, 0.5. mu.L 1492R and 1. mu.L genomic DNA. The PCR amplification program is pre-denaturation at 98 ℃ for 30s, the amplification cycle comprises denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 20s, and extension at 72 ℃ for 45s, amplification is carried out for 30 cycles, and finally extension at 72 ℃ is carried out for 2 min. After PCR amplification, the specificity is determined by 1% agarose gel electrophoresis, and then the DNA fragment is sent to Shanghai Biotechnology Limited for sequencing.
The results show that: PCR specific band of 1540bp can be obtained from separated LJ2 strain by using primer of 16S rRNA gene amplification, sequence length is 1539bp by sequencing analysis, GenBank gene accession number of the sequence is MZ 723343. The 16S rDNA sequence of strain LJ2 was compared to Pseudomonas cichorii (P.cichorii) ATCC10857 by alignment with the EzBiocloud database (www.ezbiocloud.net)TMost similarly, the sequence similarity is 99.93%. The 16S rDNA sequence of the standard strain with the genetic relationship closer to the pseudomonas chicory is obtained from the NCBI database, and the MEGA 7 software is adopted to construct a phylogenetic tree by a proximity method (Neighbor-Joining), as shown in figure 5, the result shows that the pathogenic bacteria LJ2 of the bacterial black spot of tobacco and the pseudomonas chicory (P.cichorii) ATCC10857TMost similar.
The results of the above examples show that the typical tobacco bacterial black spot disease sample is selected, the bacterial disease tissue separation method is adopted, after the separation culture is carried out for 2 days, the bacterial colony with the color from light gray to dark gray is selected, and the bacterial colony PCR identification is carried out by adopting the specific primer, so that the tobacco bacterial black spot disease can be effectively screened.
Example 2
The black spot disease sample in this example is 61 tobacco bacterial black spot disease samples collected from the yunnan lincang fengqing in 8 months in 2021.
2.1 Collection of disease specimens
Typical diseased leaves of the tobacco bacterial black spot are collected from the field, the selected diseased leaves have large irregular black spots, the surfaces of the diseased spots have no mildew layer, the edge halos are not obvious, the edges are sometimes limited by veins and side veins, and the diseased leaves are easy to occur in high-altitude areas with humidity and lower temperature. The whole leaf was placed in a sampling bag and taken back to the laboratory.
2.2 isolation of pathogenic bacteria
Washing fresh leaf scab with flowing tap water for 4 times, taking off fresh scab at the juncture of the scab with sterile blade, placing 20 scabs in sterile petri dish, sterilizing with 75% alcohol surface for 5s, sterilizing with 0.5% sodium hypochlorite for 5min, rinsing with sterile water for 3 times, and cutting sterilized scab with sterile blade into pieces of 0.5 × 0.5cm2And (3) placing the tissue blocks in a sterilized culture dish containing 20mL of sterile water, soaking for 8min, and shaking and uniformly mixing the bacterial suspension after milky bacterial liquid overflows around the tissue blocks.
2.3 pathogen culture and selection
Sucking 50 μ L of the bacterial suspension by a pipette on an NA culture medium plate, streaking by using a sterile toothpick to dilute the bacterial suspension, drying the diluted bacterial suspension on a clean bench, and placing the diluted bacterial suspension in an incubator at 30 ℃ for dark culture for 49 h.
The experimental results are as follows: suspected pathogenic bacteria were separated from 61 bacterial black spot disease samples of tobacco collected in Lincang Fengqing in 2021, the bacterial colonies were light gray in the early stage, and the bacterial colonies were dark gray after being placed in a refrigerator at room temperature or 4 ℃ for 2 days.
2.4 identification of pathogenic bacteria of bacterial black spot of tobacco
2.4.1 preparation of bacterial suspension: adding 50 mu L of sterile water into a sterilized centrifuge tube, picking out single bacterial colonies with light gray to dark gray colony centers by using sterilized toothpicks, placing the single bacterial colonies in the sterile water, and vibrating and uniformly mixing the single bacterial colonies on a vortex oscillator to prepare bacterial colony PCR bacterial suspension.
2.4.2PCR amplification
Taking 2 mu L of colony PCR bacterial suspension, and carrying out colony PCR amplification by using specific primers PcF/PcR of tobacco bacterial alternaria alternata, wherein the sequence of the primers PcF is 5'-CCGTTCATCGTCATCGACCT-3', and the sequence of the primers PcR is 5'-CCGTTCATCGTCATCGACCT-3'5'-CTGTCCCACATGATCTGGGT-3' are provided. The reaction system for colony PCR identification is 25 μ L, and the reaction system consists of 12 μ L2 XEs TaqMasterMix (Dye), 12 μ L ddH2O, 0.5. mu.L of 10. mu.M PcF, 0.5. mu.L of 10PcR and 1. mu.L of bacterial suspension; the colony PCR reaction condition is pre-denaturation at 94 ℃ for 2min, the amplification cycle comprises denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, amplification for 30 cycles, and finally extension at 72 ℃ for 2 min.
After the PCR amplification was completed, 5. mu.L PCR product was applied to a gel well of 2% agarose gel, separated at a constant pressure of 120V for 45min, and stained in EB nucleic acid staining solution containing 5. mu.g/mL for 10 min.
From 61 samples collected in Lincang Fengqing, 1 single colony with light gray to dark gray was selected for colony PCR amplification. The result shows that specific amplified bands between 750 bp and 1000bp can be amplified from the single colony, the specific single band with the size of 879bp can be amplified, 54 single colonies can be amplified to obtain the band, and the positive detection rate is 88.5%. The results show that the colony color change of the pathogenic bacteria is combined with the specific primer, so that suspected pathogenic bacteria can be distinguished specifically, and the separation accuracy of the pathogenic bacteria is improved.
2.5 verification of bacterial melasma Nicotiana in tobacco similar to example 1, 16S rDNA sequences of strains 21LCFQ39, 21LCFQ02, 21LCFQ47, 21LCFQ20, 21LCFQ05 and Pseudomonas Cichorii (P.cichorii) ATCC10857TMost similar, the sequence similarity is 99.79%, 99.50%, 99.79%, 99.64%, 99.79%, respectively. The positive colonies isolated by this method were all pathogenic bacteria of tobacco bacterial black spot, Pseudomonas chicory.
Example 3
The black spot disease samples in this example were 20 parts of tobacco bacterial black spot disease samples collected from Lincang Shuangjiang and Boshang in Yunan at 8 months in 2021.
3.1 Collection of disease specimens
Typical diseased leaves of the tobacco bacterial black spot are collected from the field, the selected diseased leaves have large irregular black spots, the surfaces of the diseased spots have no mildew layer, the edge halos are not obvious, the edges are sometimes limited by veins and side veins, and the diseased leaves are easy to occur in high-altitude areas with humidity and lower temperature. The whole leaf was placed in a sampling bag and taken back to the laboratory.
3.2 isolation of pathogenic bacteria
Washing fresh leaf scab with flowing tap water for 3 times, removing fresh scab at the juncture of the scab with sterile blade, placing 18 scabs in sterile petri dish, surface sterilizing with 75% alcohol for 5s, sterilizing with 0.5% sodium hypochlorite for 5min, rinsing with sterile water for 3 times, and cutting sterilized scabs with sterile blade into pieces of 0.5 × 0.5cm2And (3) placing the tissue blocks in a sterile culture dish containing 15mL of sterile water, soaking for 5min, and shaking and uniformly mixing the bacterial suspension after milky bacterial liquid overflows around the tissue blocks.
3.3 pathogen culture and selection
Sucking 40 μ L of the bacterial suspension by a pipette on an NA culture medium plate, marking out and diluting the bacterial suspension by using a sterile toothpick, drying the diluted bacterial suspension on a clean bench, and placing the diluted bacterial suspension in an incubator at 30 ℃ for dark culture for 36 h.
The experimental results are as follows: suspected pathogenic bacteria are separated from 8 tobacco bacterial black spot disease samples collected at lincang, the bacterial colonies are light gray at the initial stage, and the bacterial colonies are dark gray after being placed in a refrigerator at normal temperature or 4 ℃ for 2 days.
3.4 identification of pathogenic bacteria of bacterial black spot of tobacco
3.4.1 preparation of bacterial suspension: and adding 10 mu L of sterile water into a sterilized centrifuge tube, picking single bacterial colonies with the bacterial colony centers from light gray to dark gray in the sterile water by using a sterilized toothpick, and placing the bacterial colonies on a vortex oscillator to shake and mix uniformly to prepare bacterial colony PCR bacterial suspension.
3.4.2PCR amplification
Taking 2 mu L of colony PCR bacterial suspension, and carrying out colony PCR amplification by using a tobacco bacterial alternaria alternata specific primer PcF/PcR, wherein the sequence of the primer PcF is 5'-CCGTTCATCGTCATCGACCT-3', and the sequence of the primer PcR is 5'-CTGTCCCACATGATCTGGGT-3'. The reaction system for colony PCR identification is 25 μ L, and the reaction system consists of 12 μ L2 XEs TaqMasterMix (Dye), 12 μ L ddH2O, 0.5. mu.L of 10. mu.M PcF, 0.5. mu.L of 10PcR and 1. mu.L of bacterial suspension; the colony PCR reaction condition is pre-denaturation at 94 deg.C for 2min, amplification cycle comprises denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, extension at 72 deg.C for 30s, amplification for 30 cycles, and final extension at 72 deg.CStretching for 2 min.
After the PCR amplification was completed, 5. mu.L PCR product was applied to a gel well of 2% agarose gel, separated at a constant pressure of 120V for 45min, and stained in EB nucleic acid staining solution containing 5. mu.g/mL for 10 min.
From 20 samples collected in Lincang Shuangjiang and Boshang, 1 single colony with light gray to dark gray was selected and subjected to colony PCR amplification. The result shows that a specific amplification band between 750 and 1000bp can be amplified from the single colony, the specific size is 897bp, only 1 single colony has no amplification band, and the positive detection rate is 95.00%. The results show that the colony color change of the pathogenic bacteria is combined with the specific primer, so that suspected pathogenic bacteria can be distinguished specifically, and the separation accuracy of the pathogenic bacteria is improved.
3.5 verification of bacterial black spot pathogen of tobacco in the same manner as in example 1, the 16S rDNA sequences of the strains LCSJ12, LCBS23 and Pseudomonas cichorii (P.cichororii) ATCC10857 were compared with each other by EzBiocloud database (www.ezbiocloud.net)TMost similar, the sequence similarity was 99.86%, 99.79%, respectively. The separated LCSJ12 and LCBS23 are the pathogenic bacteria of tobacco bacterial black spot and pseudomonas chicory.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (9)
1. A method for separating and identifying pathogenic bacteria of bacterial black spot of tobacco is characterized by comprising the following steps: the method comprises the following steps:
s1: collecting disease samples: collecting diseased leaves with tobacco bacterial black spot disease spots in the field, putting the diseased leaves into a sampling bag, and bringing the sampling bag back to a laboratory;
s2: and (3) pathogenic bacteria separation: washing the tobacco bacterial black spot disease leaves with disease spots in the step S1 for 3-5 times by using flowing tap water, taking a plurality of fresh disease spots at disease-health junctions, and rinsing after disinfecting in a sterile culture dish; cutting the rinsed lesion spots into a plurality of tissue blocks, placing the tissue blocks in a sterilized culture dish containing 10-20mL of sterile water, soaking for 5-10min, and shaking the culture dish to uniformly mix bacterial liquid after milky bacterial liquid overflows around the tissue blocks to obtain bacterial suspension;
s3: and (3) pathogenic bacteria culture and screening: sucking 30-50 mu L of the bacterial suspension obtained in the step S2 into an NA culture medium plate by using a pipettor, streaking and diluting the bacterial suspension by using a sterile toothpick, and drying the diluted bacterial suspension by placing the diluted bacterial suspension on an ultra-clean workbench; drying, placing in an incubator at 28-30 ℃ for dark culture for 36-49h, culturing for 23-25h to form a plurality of small single colonies, culturing for 47-49h to obtain light gray single colonies, and placing the light gray single colonies in an environment at normal temperature or 4 ℃ for 2-5d to obtain dark gray single colonies;
s4: preparing bacterial colony PCR bacterial suspension: adding 10-100 mu L of sterile water into a sterilized centrifuge tube, picking a dark gray single bacterial colony in the step S3 by using a sterilized toothpick, placing the bacterial colony in a vortex oscillator, and vibrating and uniformly mixing to prepare bacterial colony PCR bacterial suspension;
s5: PCR amplification and identification: taking 0.5-2 mu L of bacterial colony PCR bacterial suspension in the step S4, carrying out bacterial colony PCR amplification by using tobacco bacterial alternaria alternata specific primers PcF and PcR, and obtaining a PCR product after the PCR amplification is finished; taking 5-10 mu LPCR product, loading the product into a gel hole of 0.8-1.5% agarose gel, separating for 30-45min under the constant pressure of 120-145V, and staining for 10-20min in EB nucleic acid staining solution containing 5-10 mu g/mL; placing the mixture on a gel imager to obtain the separation condition of a PCR product, wherein if the mixture is the tobacco bacterial macula nigra, a specific amplification strip exists between 750 bp and 1000 bp;
s6: 16S rRNA gene amplification and validation: and (3) shaking the bacteria of which the colony PCR amplification is positive for 24h by using an LB culture medium, extracting genome DNA by using a bacteria genome DNA extraction kit, carrying out PCR amplification by using universal primers 27F and 1492R for 16S rRNA gene amplification, sequencing the amplified product, and analyzing the sequencing result to determine the pathogenic bacteria.
2. The method for isolating and identifying a pathogen of tobacco bacterial black spot according to claim 1, wherein the disease spots on the diseased leaves in step S1 are irregular large black spots, no mold layer is formed on the surface of the black spots, halos are not evident at the edges of the black spots, and the edges of the black spots are restricted by veins or collateral veins.
3. The method for separating and identifying pathogenic bacteria of bacterial black spot of tobacco according to claim 1, wherein more than 10 fresh spots are collected in step S2; the disinfection is to disinfect the surface of the steel tube by using 75% alcohol for 5s and then disinfect the steel tube by using 0.5% sodium hypochlorite for 5 min; the rinsing is 3 times of rinsing with sterile water; the tissue block has a size of 0.5 × 0.5cm2。
4. The method for separating and identifying pathogenic bacteria of bacterial black spot of tobacco according to claim 1, wherein the NA medium plate of step S3 is prepared by: respectively weighing 18g of nutrient broth and 15.0g of agar, adding distilled water to a constant volume of 1L, autoclaving at 121 ℃ for 20min, placing in a 55 ℃ water bath kettle, adding 23-28mL of culture medium into each sterile culture dish when the temperature of the culture medium is reduced to about 60 ℃, and condensing to obtain an NA plate.
5. The method for isolating and identifying bacterial melasma pathogenic bacteria of tobacco according to claim 1, wherein in step S5, primer PcF has a sequence of 5'-CCGTTCATCGTCATCGACCT-3' and primer PcR has a sequence of 5'-CTGTCCCACATGATCTGGGT-3'.
6. According to the claimsClaim 5, wherein the reaction system of PCR identification in the step S5 is 25 μ L, and the reaction system comprises 12 μ L of 2 × Es TaqMasterMix (Dye) and 12 μ L ddH2O, 0.5 muL of 10 muM PcF, 0.5 muL of 10PcR and 0.5-2.0 muL of bacterial suspension; the colony PCR reaction condition is pre-denaturation at 94 ℃ for 2min, the amplification cycle comprises denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, the amplification cycle comprises 30 cycles, and finally extension at 72 ℃ for 2min, and the PCR product is stored at 16 ℃ or directly used for agarose gel electrophoresis.
7. The method according to claim 6, wherein there is a specific amplification band between 750 and 1000bp, specifically a band at 897bp, in step S5.
8. The method for isolating and identifying bacterial melasma pathogenic bacteria of tobacco according to claim 1, wherein the PCR reaction system of the 16S rRNA gene in step S6 is 16 μ L ddH2O, 3. mu.L dNTP MIX, 5. mu. L Q5 Reaction Buffer, 0.3. mu. L Q5 DNA Polymerase, 0.5. mu.L 27F, 0.5. mu.L 1492R and 0.5-2. mu.L genomic DNA, the PCR amplification program is pre-denaturation at 98 ℃ for 30s, the amplification cycle comprises denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 20s, extension at 72 ℃ for 45s, amplification for 30 cycles, and finally extension at 72 ℃ for 2 min.
9. The method for isolating and identifying a pathogen of tobacco bacterial black spot according to claim 1, wherein said pathogen identified in step S6 is Pseudomonas chicory Pseudomonas pseudomonias cichorii.
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