Separation, purification and identification method of cabbage black spot pathogenic bacteria

Abstract

The invention relates to the technical field of cabbage pathogenic bacteria detection, in particular to a cabbage black spot pathogenic bacteria separation, purification and identification method which comprises the following steps: collecting leaf disease samples; separating pathogenic bacteria; purifying pathogenic bacteria; expanding propagation of pathogenic bacteria; and identifying pathogenic bacteria (morphological identification and molecular biological identification). Compared with an existing separation, purification and identification method of the cabbage black spot pathogenic bacteria, the method stands out due to the simplicity and convenience of operation, and the separation and purification period is remarkably shortened. Meanwhile, by combining dual identification means of morphology and molecular biology, the cabbage black spot pathogenic bacteria are accurately identified, so that the identification process of the pathogenic bacteria is greatly accelerated, and qualitative leap is realized on the accuracy of the identification result. The method also has important significance in promoting the disease-resistant breeding research of the cabbages and the implementation of a comprehensive prevention and control strategy, and shows extremely wide application potential and prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of cabbage pathogen detection, and particularly relates to a method for separating, purifying and identifying cabbage black spot pathogens. Background Art

[0002] Currently, the pathogens reported to cause black spot disease of cabbage include Alternaria brassicicola, Alternaria brassicae, and Alternaria alternata. Among them, Alternaria brassicicola is the pathogen that causes black spot disease of cabbage with the highest frequency.

[0003] Because the pathogens that cause black spot disease in cabbage are complex and diverse, and there are often subtle morphological differences between different species, relying solely on traditional morphological characteristics for identification is not only time-consuming and laborious, requiring multiple steps such as tedious sample processing, microscopic observation, and feature comparison, but also easily affected by human factors and environmental conditions during the identification process, resulting in certain inaccuracies. In contrast, the simultaneous use of molecular biological identification methods can greatly improve the accuracy and efficiency of identification. By extracting the genetic material of the pathogen and analyzing it using specific gene sequences, the pathogen's type can be quickly and accurately identified, providing strong support for disease prevention and control.

[0004] To this end, in response to the problems raised in the above background technology, those skilled in the art have proposed a method for separating, purifying and identifying the pathogen of cabbage black spot disease. Summary of the Invention

[0005] In view of this, the present invention provides a method for separating, purifying and identifying the pathogen of cabbage black spot disease.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for separating, purifying and identifying the pathogen of cabbage black spot disease, which comprises the following steps:

[0008] (1) Collecting cabbage plants with typical symptoms of black spot disease and collecting the diseased leaves for later use;

[0009] (2) Isolation and purification of pathogens

[0010] A1: Rinse the diseased leaves and cut out the diseased tissue samples;

[0011] A2: Sterilize and air-dry the tissue sample, then place it in PDA solid culture medium and incubate it in the dark at 25-28°C until hyphae grow around the tissue sample.

[0012] A3: Transfer the hyphae at the edge of the tissue sample into a new culture medium. After new hyphae are cultured, repeat this step 3 to 6 times until a single colony is obtained, thus obtaining the purified pathogen.

[0013] (3) Propagation of the pathogen causing black spot disease in cabbage

[0014] After the purified pathogens are cultured to produce new mycelia using the streak method or the bacterial block method, the new mycelia are transferred to a new PDA solid culture medium and cultured in the dark to obtain the expanded pathogens;

[0015] (4) Morphological identification of the pathogen causing black spot disease of cabbage

[0016] Observe and record the colony morphology, spore morphology, and conidia chain morphology of the pathogens expanded in step (3);

[0017] (5) Molecular biological identification of the pathogen causing black spot disease in cabbage

[0018] B1: Extract DNA of the pathogen of black spot disease of cabbage, perform PCR amplification on the ITS1, TEF1, and GAPDH genes of the test strain, and obtain amplified products;

[0019] B2: After the amplified product is sequenced, the complete strain gene sequence is searched for homologous sequences by BLAST on the NCBI website, multiple sequence alignment is performed using MAFFT, and a phylogenetic tree is constructed using FastTree to identify the species of the pathogen.

[0020] Preferably, the dark culture time in step A2 is 2 to 3 days.

[0021] Preferably, the culture method in step A3 is dark culture, the culture temperature is 25-28° C., and the culture time is 8-16 hours.

[0022] Preferably, the streak method comprises the following steps: adding sterile water to a culture medium containing purified pathogens, scraping conidia from the surface of the colony with a sterile coating stick, filtering the conidia with two layers of sterilized lens paper, streaking the PAD culture medium with a sterile inoculating loop, and after the spores grow hyphae, picking the hyphae and placing them in the center of the PAD culture medium, which is then placed in a constant temperature incubator for dark culture.

[0023] Preferably, the specific steps of the bacterial block method are: cutting a mycelium block of 0.5 cm in size from the edge of the purified pathogen colony, placing it in five positions of top, bottom, left, right and center in a new PDA culture medium, waiting for new mycelium to grow out of the bacterial block, picking the mycelium and transferring it to the center position of the new PDA culture medium, and placing it in a constant temperature incubator for dark culture.

[0024] Preferably, the dark culture temperature in step (3) is 25-28° C. and the time is 7-15 days.

[0025] Preferably, the nucleotide sequences of the primers used for PCR amplification in step B1 are shown as SEQ ID NOs. 1 to 6.

[0026] Preferably, the reaction system for PCR amplification in step B1 is 10 to 40 μL, and its specific components are:

[0027] 5-20 μL 2× Rapid Tag MasterMix, 1-2 μL genomic DNA, 0.5-2.5 μL each of upstream and downstream primers, 3-14 μL ddH2O.

[0028] More preferably, the total reaction volume of the PCR amplification in step B1 may be 10, 20, or 40 μL;

[0029] The specific components of the 10 μL PCR amplification system are:

[0030] 5 μL 2× Rapid Tag MasterMix, 1 μL genomic DNA, 0.5 μL each of upstream and downstream primers, 3 μL ddH2O.

[0031] The specific components of the 20 μL PCR amplification system are:

[0032] 10 μL 2× Rapid Tag Master Mix, 2 μL genomic DNA, 1 μL each of upstream and downstream primers, and 6 μL ddH2O.

[0033] The specific components of the 40 μL PCR amplification system are:

[0034] 20 μL 2× Rapid Tag MasterMix, 1 μL genomic DNA, 2.5 μL each of upstream and downstream primers, and 14 μL ddH2O.

[0035] Contains at least the following beneficial technical effects:

[0036] 1. The present invention finally identified the pathogen of cabbage black spot disease as Alternaria brassicicola through separation, purification, propagation, morphological identification and molecular biological identification.

[0037] 2. The present invention uses PCR amplification and molecular sequencing, and the sequencing results are searched for homologous sequences on the NCBI website by BLAST. Multiple sequence alignment is performed using MAFFT, and a phylogenetic tree is constructed using FastTree to identify the species of the pathogen. This method achieves rapid isolation and accurate identification of the pathogen of cabbage black spot disease, which is more efficient and more accurate than traditional isolation and identification methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is an operational flow chart of the method for separating, purifying and identifying the pathogen of cabbage black spot disease of the present invention;

[0039] Figure 2 Schematic diagram of a typical cabbage black spot disease-infected plant and collected diseased leaves of the present invention, wherein A is the diseased plant and B is the diseased leaf;

[0040] Figure 3 The black spot pathogen isolated by the present invention is a colony grown around leaf tissue in a solid PDA culture medium;

[0041] Figure 4 The morphological identification diagram of the black spot pathogen of cabbage after propagation of the present invention; wherein A is a colony morphology diagram, B is a spore morphology diagram, and C is a conidia chain diagram;

[0042] Figure 5 This is a phylogenetic tree of the black spot pathogen Alternaria brassicicola constructed based on the ITS gene of the present invention;

[0043] Figure 6 This is a phylogenetic tree of the black spot pathogen Alternaria brassicicola constructed based on the TEF1 gene of the present invention;

[0044] Figure 7 The present invention shows a phylogenetic tree of the black spot pathogen Alternaria brassicicola constructed based on the GAPDH gene. DETAILED DESCRIPTION

[0045] Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without creative work shall fall within the scope of protection of the present invention. The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are merely illustrative and do not limit the present invention in any way.

[0046] The solid PDA culture medium described in the following examples is prepared as follows: The product (Oxoid, Catalog No. CM0139) is prepared according to the instructions. Finally, it is autoclaved at 121°C for 20 minutes and then placed in a 55°C oven. Once the culture medium temperature drops to approximately 60°C, 25 mL of the culture medium is added to each sterile culture dish and condensed to produce the solid PDA culture medium.

[0047] Example 1

[0048] A method for separating, purifying and identifying the pathogen of cabbage black spot disease, the method comprising the following steps:

[0049] (1) Collecting leaf samples of black spot disease in cabbage

[0050] In the main cabbage producing areas of Chongqing and Inner Mongolia, cabbage plants with typical symptoms of black spot disease were selected (see Figure 1 ), take photos and collect diseased leaves (see Figure 2 ), place in a fresh-keeping bag, and bring back to the laboratory for use.

[0051] (2) Isolation of the pathogen causing black spot disease of cabbage

[0052] Prepare solid PDA medium in advance.

[0053] Wash the surface of the diseased cabbage sample collected in step (1) with sterile water and dry it for later use;

[0054] At the junction of the diseased and healthy parts, 5 mm × 5 mm pieces of diseased leaf tissue were cut with a sterile scalpel and numbered 1-10;

[0055] The experiment was performed in a clean bench: tissue samples numbered 1-10 were sequentially immersed in 3% sodium hypochlorite solution for 40 seconds, then sterilized in 75% alcohol for 30 seconds, and finally rinsed three times in sterile water. Sterile forceps were used to place the tissue blocks on sterile filter paper and air-dried them for later use.

[0056] After drying, use a sterile scalpel to split the tissue blocks into two and place them in solid PDA culture medium. Place five small tissue blocks evenly on the top, bottom, left, right, and middle of each dish, record the numbers, and seal with parafilm.

[0057] The tissue blocks were placed in a 28°C constant temperature incubator for dark culture for 2 days. After 12 hours, the growth of fungal hyphae around the tissue blocks was observed every 6 hours.

[0058] (3) Purification of the pathogen causing black spot disease of cabbage

[0059] After hyphae grow around the tissue block separated in step (2), use a sterile scalpel or inoculation loop to pick up the hyphae near the tissue block and transfer them to fresh PDA culture medium. Record the numbers in sequence, for example, 1-1, 6-1 (the third transfer can be recorded as 9-3, 5-3), place them in a 28°C constant temperature incubator and culture them in the dark for 12 hours. Observe the growth of the strain regularly.

[0060] When new mycelium grows, repeat the above steps and pick the new mycelium at least 3 times;

[0061] If the strain grows into non-single colonies, the above purification steps should be repeated several times until pure colonies are obtained (see Figure 3 ).

[0062] (4) Propagation of the pathogen causing black spot disease in cabbage

[0063] The propagation of the pathogen of cabbage black spot disease, the purified pathogens need to be propagated, each plate is propagated 10, including the streak method and the bacterial block method. The specific steps are as follows:

[0064] ① Streak method: Add sterile water to the pathogen purified in step (3), scrape the conidia on the surface of the culture medium with a sterile coating stick, filter the conidia with two layers of sterile lens paper, and streak on a new PAD culture medium with a sterile inoculating loop. After the spores grow hyphae, use a sterile scalpel to take a small piece of agar containing hyphae and place it in the center of the new PAD culture medium. Place it in a 28°C constant temperature incubator and culture it in the dark for 15 days.

[0065] ② Mushroom block method: Use a sterilized scalpel to take a 0.5 cm mycelium block from the edge of the colony of the pathogen purified in step (3), and inoculate it evenly in the top, bottom, left, right and middle positions of fresh PDA. After new mycelia grow on the block, pick out the new mycelia and transfer it to fresh PDA culture medium. Place it in a 28°C constant temperature incubator and culture it in the dark for 15 days.

[0066] (5) Morphological identification of the pathogen causing black spot disease of cabbage

[0067] Morphological identification includes colony morphology, spore morphology, and conidia chain morphology. The specific steps are as follows:

[0068] The pathogenic bacteria plate expanded in step (3) was cultured in the dark for 15 days and then used.

[0069] ① Colony morphology: observe the size, color and hyphae of the colony and take photos (see Figure 4 A).

[0070] ② Spore morphology: Use sterile water and a coating stick to scrape the conidia from the surface of the bacterial plate, filter the conidia with two layers of lens paper, and observe the morphological characteristics of the conidia under an optical microscope (see Figure 4 B).

[0071] ③ Conidia chain morphology: Touch the surface of the colony with a transparent tape, fix the tape on the slide with a drop of sterile water, and observe the morphological characteristics of the conidia chain under an optical microscope (see Figure 4 C).

[0072] Depend on Figure 4The colony morphology of the pathogen of cabbage black spot disease is concentric whorls with a dark brown center and a uniform gray-brown periphery. Aerial hyphae are sparse, and conidia are dense. Conidia are inverted club-shaped, with no or indistinct beaks, and have a spore diameter of (2.16-8.39) × (1.67-2.63) μm. The number of transverse septa ranges from 1 to 3, and the number of mediastinums ranges from 0 to 1. Conidial chains are short, dwarf tree-like branches, exhibiting typical morphological characteristics of Alternaria brassicicola.

[0073] If, after the above steps, the observed colony morphology, spore morphology and conidia chain morphology do not have the above typical morphological characteristics, it can be determined that the identified strain is not the pathogen of cabbage black spot disease.

[0074] (6) Molecular biological identification of the pathogen causing black spot disease in cabbage

[0075] ① Extraction of pathogen DNA, the specific steps are as follows:

[0076] Take the pathogen plate expanded in step (3) and the purified pathogens and extract DNA. In a clean bench, use a sterilized scalpel or inoculation loop to pick up 0.3g of bacterial mass and place it in a 2ml centrifuge tube, and record the number.

[0077] Add 200 μL of lysis solution (0.5 mol / L NaOH liquid) and two steel balls (2 mm diameter);

[0078] Place the sample in a proofing machine and quickly proof until it becomes a paste (50 Hz, 2 min), let it stand for 10 min, and then centrifuge it (12000 rpm, 10 min);

[0079] Add 100 μL of neutralizing solution (100 mmol / L Tris-HCl and 1 mmol / L EDTA liquid) to a new 1.5 ml centrifuge tube, transfer 20 μL of supernatant to the neutralizing solution, record the number and store in a -20°C refrigerator for long-term storage.

[0080] ②PCR amplification and identification, the specific steps are as follows:

[0081] Using pathogen DNA as template, target gene primers were used to amplify the ITS1, TEF1, and GAPDH genes of the test strains by PCR.

[0082] PCR amplification system (40 μL):

[0083] 20 μL 2× Rapid Tag MasterMix, 1 μL genomic DNA, 2.5 μL each of upstream and downstream primers, 14 μL ddH2O;

[0084] Primer sequences are shown in Table 1:

[0085] Table 1 PCR amplification primers

[0086]

[0087] The PCR reaction program is shown in Table 2:

[0088] Table 2 PCR reaction procedure

[0089]

[0090] After electrophoresis on a 1.5% agarose gel, the amplified product was sequenced to obtain the pathogen sequence, and the number was recorded throughout the process.

[0091] ③ The sequencing results were compared with BLAST in GenBank, and then the phylogenetic tree was constructed using MEGA software to identify the species of the pathogen (see Figures 5-7 ).

[0092] Finally, after double identification in steps (5) to (6), the pathogen of cabbage black spot disease was accurately identified as Alternaria brassicicola. Referring to Koch's postulates, the purified pathogen was again used to prepare a spore suspension (the purified PDA black spot pathogen plate was added with sterile water, and conidia on the surface of the culture medium were scraped with a coating stick, and the conidia were filtered with two layers of lens paper. The concentration of the spore suspension was determined using a hemocytometer and adjusted to 1×10 5 Spores / ml) were artificially inoculated on cabbage leaves at the four-leaf and one-heart stage. After obvious disease symptoms appeared, the above steps (1) to (6) were repeated to further confirm that the isolated pathogen was Alternaria brassicicola.

[0093] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A method for separating, purifying and identifying the pathogen of cabbage black spot disease, characterized in that: The method comprises the following steps: (1) Collecting cabbage plants with typical symptoms of black spot disease and collecting the diseased leaves for later use; (2) Isolation and purification of pathogens A1: Rinse the diseased leaves and cut out the diseased tissue samples; A2: Sterilize and air-dry the tissue sample, then place it in PDA solid culture medium and incubate it in the dark at 25-28°C until hyphae grow around the tissue sample. A3: Transfer the hyphae at the edge of the tissue sample into a new culture medium. After new hyphae are cultured, repeat this step 3 to 6 times until a single colony is obtained, thus obtaining the purified pathogen. (3) Propagation of the pathogen causing black spot disease in cabbage After the purified pathogens are cultured to produce new mycelia using the streak method or the bacterial block method, the new mycelia are transferred to a new PDA solid culture medium and cultured in the dark to obtain the expanded pathogens; (4) Morphological identification of the pathogen causing black spot disease of cabbage Observe and record the colony morphology, spore morphology, and conidia chain morphology of the pathogens expanded in step (3); (5) Molecular biological identification of the pathogen causing black spot disease in cabbage B1: Extract DNA of the pathogen of black spot disease of cabbage, perform PCR amplification on the ITS1, TEF1, and GAPDH genes of the test strain, and obtain amplified products; B2: After the amplified product is sequenced, the complete strain gene sequence is searched for homologous sequences by BLAST on the NCBI website, multiple sequence alignment is performed using MAFFT, and a phylogenetic tree is constructed using FastTree to identify the species of the pathogen.

2. The method for separating, purifying and identifying the pathogen of cabbage black spot according to claim 1, wherein: The dark culture time in step A2 is 2 to 3 days.

3. The method for separating, purifying and identifying the pathogen of cabbage black spot according to claim 1, wherein: The culture method in step A3 is dark culture, the culture temperature is 25-28° C., and the culture time is 8-16 hours.

4. The method for separating, purifying and identifying the pathogen of cabbage black spot according to claim 1, wherein: The specific steps of the streak method are as follows: sterile water is added to the culture medium containing the purified pathogen, conidia are scraped from the surface of the colony with a sterile coating stick, the conidia are filtered through two layers of sterilized lens paper, and a sterile inoculating loop is used to streak on the PAD culture medium. After the spores grow into hyphae, the hyphae are picked and placed in the center of the PAD culture medium, and then placed in a constant temperature incubator for dark culture.

5. The method for separating, purifying and identifying the pathogen of cabbage black spot according to claim 1, wherein: The specific steps of the bacterial block method are as follows: cutting a 0.5 cm mycelium block from the edge of a purified pathogen colony, placing it in five positions (upper, lower, left, right, and center) in a new PDA culture medium, waiting for new mycelium to grow, picking the mycelium and transferring it to the center of the new PDA culture medium, and placing it in a constant temperature incubator for dark culture.

6. The method for separating, purifying and identifying the pathogen of cabbage black spot according to claim 4 or 5, characterized in that: The dark culture temperature in step (3) is 25-28° C. and the time is 7-15 days.

7. The method for separating, purifying and identifying the pathogen of cabbage black spot according to claim 1, wherein: The nucleotide sequences of the primers used for PCR amplification in step B1 are shown in SEQ ID NOs. 1 to 6.

8. The method for separating, purifying and identifying the pathogen of cabbage black spot disease according to claim 1, characterized in that: The reaction system for PCR amplification in step B1 is 10-40 μL, and its specific components are: 5-20 μL 2× Rapid Tag MasterMix, 1-2 μL genomic DNA, 0.5-2.5 μL each of upstream and downstream primers, 3-14 μL ddH2O.

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