Separation, purification and identification method of tea cake disease pathogenic bacteria

Abstract

The invention discloses a method for separating, purifying and identifying tea cake disease pathogenic bacteria, which is characterized by comprising the following steps of: firstly collecting disease samples, then pretreating diseased leaves, then separating the pathogenic bacteria, then selecting hyphae for purifying and culturing, then respectively verifying the pathogenic bacteria and extracting DNA (deoxyribonucleic acid) of the pathogenic bacteria, and finally carrying out PCR (polymerase chain reaction) amplification and identification. According to the method, the ejection characteristic of mature basidiospore is fully utilized, the diseased tissue is directly fixed on the culture dish cover for isolated culture, the probability of contamination of endophyte and influence of infectious microbes is extremely low, the culture efficiency is improved, and tea cake disease pathogenic bacteria can be ensured; pCR amplification confirmation is carried out on the rDNA-ITS region of the pathogenic bacteria through universal primers ITS1 and ITS4 in a genome transcription spacer region, so that the selectivity of a target strain is enhanced, and the pathogenic bacteria can be rapidly identified.

Description

Technical Field

[0001] The invention relates to a method for separating, purifying and identifying pathogenic bacteria, in particular to a method for separating, purifying and identifying pathogenic bacteria of tea cake disease, and belongs to the technical field of crop plant protection. Background Art

[0002] Tea cake disease is an important disease that harms the tender shoots and leaves of tea trees. In recent years, with the adjustment of industrial structure and the continuous improvement of planting level, the occurrence of tea cake disease has gradually worsened, and the annual production is reduced by 20%-30% due to the occurrence of tea cake disease. The dry tea made from diseased leaves tastes abnormally bitter, the bottom of the leaves is dark, and the quality is poor, which will seriously affect the economic benefits of tea. The successful separation and acquisition of target pathogens from diseased tissues is a key link in plant pathology research and disease prevention and control. However, the existing technology is difficult to separate and purify the pathogens of tea cake disease, which is easily affected by miscellaneous bacteria and cannot quickly and accurately obtain the pathogens of tea cake disease. The difficulties and complexities of separating and purifying the pathogens of tea cake disease using existing technologies are mainly reflected in the following aspects: Difficulty in separation: The pathogen of tea cake disease is Exobasidiomycetes, which belongs to the Basidiomycetes. This pathogen grows very slowly in nature. During the long growth cycle, tea leaves are easily infected by other fungi, which increases the difficulty of separation. Environmental factors: Tea cake disease is different from general tea tree diseases. It mainly occurs on the tender leaves and stems of tea trees. The tissues in these parts are relatively fragile. At the same time, the formation and development of lesions are greatly affected by environmental factors and require low temperature and high humidity. When the temperature exceeds 30°C, the survival and spread of pathogens will be affected, thus affecting the separation effect. Complexity of separation technology: The isolation of the pathogen of tea cake disease is different from that of common pathogenic fungi. The isolation of this fungus requires full use of the characteristics of mature basidiospore ejection, rather than simply placing the diseased tissue on the culture medium for cultivation. These technologies require operators to have a certain professional knowledge background. In addition, the growth state of the pathogen of tea cake disease has a great influence on the separation. The ejection ability of basidiospores at different stages is different. These factors increase the difficulty of separation.

[0003] Difference in separation culture medium: The germination rate of the pathogen of tea cake disease on ordinary PDA culture medium is low. When isolating the damaged exobasidiomycetes, tea juice needs to be added to the PDA culture medium, which is cumbersome and has poor separation effect.

[0004] There has always been a lack of a method that has high culture efficiency, can avoid the influence of foreign bacteria to the greatest extent, has high separation and purification efficiency, and is easy to identify to solve the above technical problems. Summary of the invention

[0005] The purpose of the present invention is to provide a method for separating, purifying and identifying tea cake disease pathogens, which is convenient to operate, improves the culture efficiency, has extremely low probability of contamination by endophytes and being affected by miscellaneous bacteria, greatly reduces the difficulty of separating and purifying tea cake disease pathogens, has high separation and purification efficiency and is easy to identify, in view of the defects and shortcomings of the prior art, such as great difficulty in separating and purifying tea cake disease pathogens, low germination rate, susceptibility to the influence of miscellaneous bacteria, cumbersome operation, poor separation effect and inability to quickly and accurately obtain tea cake disease pathogens.

[0006] To achieve the above-mentioned purpose, the technical solution of the present invention is: a method for separating, purifying and identifying the pathogenic bacteria of tea cake disease, comprising the following steps: S1. Disease sample collection: Collect diseased leaves from the tea garden, put them into a collection bag, store them at low temperature, and bring them back to the laboratory; S2. Pretreatment of diseased leaves: Select some relatively tender, vigorous diseased leaves that are not contaminated by other fungi and are in the early stage of the disease from the picked diseased leaves, rinse them with running tap water for 5-10 minutes, cut 5mm×5mm fresh diseased spot tissue at the junction of diseased and healthy leaves with a sterile scalpel on a sterile operating table, disinfect them in a sterile culture dish and rinse them; S3. Isolation of pathogens: Stick the rinsed leaves with the back side facing down on the PDA plate cover, and incubate them in an incubator at 25 degrees Celsius with light, allowing the spores to fall freely; S4. Pick hyphae for purification and culture: Using the characteristics of mature basidiospore ejection, directly fix the diseased tissue on the cover of the culture dish for separation and culture. After culturing for about 20 days, when the hyphae are white velvety and slightly shiny, gently pick the hyphae tip at the edge of the colony and move it to the fresh tea juice PDA plate for purification and culture; S5. Pathogen verification: Punch the purified tea cake disease pathogen obtained, inoculate it on the young leaves of tea plants, and culture it under heat and moisture preservation until the symptoms of tea cake disease appear and a white hymenium is produced, and then re-isolate the tea cake disease pathogen strain from the diseased detached leaf spots; S6. Pathogen DNA extraction: Use the Fungal Genomic DNA Rapid Extraction Kit to extract DNA, add an appropriate volume of sterile double distilled water or TE buffer to precipitate, and store at 4°C for later use or at -20°C for long-term storage; S7, PCR amplification: PCR amplification of the pathogen rDNA-ITS region was performed using universal primers ITS1 and ITS4 of the genomic transcribed spacer region; S8. PCR identification: The PCR amplification product was electrophoresed on a 1.0% agarose gel in 0.5×TBE buffer. After EB staining, it was placed on a gel imager to obtain the separation of the PCR products. If it is the pathogen of tea cake disease, there will be a specific amplification band near 540 bp.

[0007] Furthermore, in the step S2, the sterile culture dish is disinfected with 75% alcohol for 3 times, each time for 30 seconds; and after disinfection, the dish is rinsed with sterile water for 3 times, each time for 1 minute.

[0008] Furthermore, in step S4, the temperature of the incubator is set to 15-32°C.

[0009] Furthermore, the optimum temperature of the incubator in step S4 is 25°C.

[0010] Furthermore, the reaction system for PCR amplification in step S7 is 25 μL: PCR Buffer 2.5 μL, primers 1 μL each, DNA template 1 μL, Taq DNA polymerase 0.4 μL, dNTP 0.5 μL, and ddH2O is used to make up to 25 μL.

[0011] Furthermore, the PCR amplification reaction procedure in the S7 step is as follows: the first cycle is pre-denaturation at 94°C for 4 minutes; then denaturation at 94°C for 45 seconds, annealing at 54°C for 45 seconds, and extension at 54°C for 45 seconds, for a total of 35 cycles; and finally extension at 72°C for 10 minutes.

[0012] Furthermore, the voltage of electrophoresis in step S8 is 4-5 V / cm.

[0013] The beneficial effects of the present invention are: 1. The present invention makes full use of the characteristics of mature basidiospore ejection, directly fixes the diseased tissue on the culture dish cover for separation and culture, and the probability of contamination by endophytes and the influence of miscellaneous bacteria is extremely low, thereby improving the culture efficiency and ensuring that it is the pathogen of tea cake disease.

[0014] 2. The present invention uses the universal primers ITS1 and ITS4 of the genomic transcribed spacer region to perform PCR amplification and confirmation on the rDNA-ITS region of the pathogen, thereby enhancing the selectivity of the target strain and being able to quickly identify the pathogen.

[0015] 3. The present invention is easy to operate, greatly reduces the difficulty of separating and purifying the pathogenic bacteria of tea cake disease, has high separation and purification efficiency, can accurately obtain the target pathogen, and lays a solid foundation for subsequent research. General scientific and technological personnel can also easily master this method. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 These are the symptoms of tea cake disease in tea gardens at different stages.

[0017] Figure 2 It is a front morphological diagram of the bacterial colony of the present invention.

[0018] Figure 3 It is the reverse morphological diagram of the bacterial colony of the present invention.

[0019] Figure 4 This is a test table of the difference significance of the effects of different temperatures on the spore germination rate of the present invention.

[0020] Figure 5 The invention compares the results of obtaining pathogens by using conventional separation method and spore ejection method for specimens from different sources.

[0021] Figure 6 This is a diagram of an in vitro inoculation test of the pathogenic bacteria of tea cake disease of the present invention.

[0022] Figure 7 It is an electrophoresis observation diagram of the rDNA-ITS PCR amplification result of the tea cake disease pathogen of the present invention.

[0023] Figure 8 It is the sequencing comparison result of the PCR products of the present invention. DETAILED DESCRIPTION

[0024] The present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0025] See also Figures 1 to 8 The present invention provides a method for separating, purifying and identifying the pathogenic bacteria of tea cake disease, which is characterized by comprising the following steps: S1. Disease sample collection: Collect diseased leaves from the tea garden, put them into a collection bag, store them at low temperature, and bring them back to the laboratory; S2. Pretreatment of diseased leaves: Select some relatively tender, vigorous diseased leaves that are not contaminated by other fungi and are in the early stage of the disease from the picked diseased leaves, rinse them with running tap water for 5-10 minutes, cut 5mm×5mm fresh diseased spot tissue at the junction of diseased and healthy leaves with a sterile scalpel on a sterile operating table, disinfect them in a sterile culture dish and rinse them; S3. Isolation of pathogens: Stick the rinsed leaves with the back side facing down on the PDA plate cover, and incubate them in an incubator at 25 degrees Celsius with light, allowing the spores to fall freely; S4. Pick hyphae for purification and culture: Using the characteristics of mature basidiospore ejection, directly fix the diseased tissue on the cover of the culture dish for separation and culture. After culturing for about 20 days, when the hyphae are white velvety and slightly shiny, gently pick the hyphae tip at the edge of the colony and move it to the fresh tea juice PDA plate for purification and culture; S5. Pathogen verification: Punch the purified tea cake disease pathogen obtained, inoculate it on the young leaves of tea plants, and culture it under heat and moisture preservation until the symptoms of tea cake disease appear and a white hymenium is produced, and then re-isolate the tea cake disease pathogen strain from the diseased detached leaf spots; S6. Pathogen DNA extraction: Use the Fungal Genomic DNA Rapid Extraction Kit to extract DNA, add an appropriate volume of sterile double distilled water or TE buffer to precipitate, and store at 4°C for later use or at -20°C for long-term storage; S7, PCR amplification: PCR amplification of the pathogen rDNA-ITS region was performed using universal primers ITS1 and ITS4 of the genomic transcribed spacer region; S8. PCR identification: The PCR amplification product was electrophoresed on a 1.0% agarose gel in 0.5×TBE buffer. After EB staining, it was placed on a gel imager to obtain the separation of the PCR products. If it is the pathogen of tea cake disease, there will be a specific amplification band near 540 bp.

[0026] In the step S2, the sterile culture dish is disinfected with 75% alcohol for 3 times, each time for 30 seconds; during rinsing after disinfection, it is rinsed with sterile water for 3 times after each alcohol disinfection, each time for 1 minute.

[0027] In the step S4, the temperature of the incubator is set to 15-32°C.

[0028] The optimum temperature of the incubator in step S4 is 25°C.

[0029] The reaction system for PCR amplification in step S7 is 25 μL: PCR Buffer 2.5 μL, primers 1 μL each, DNA template 1 μL, Taq DNA polymerase 0.4 μL, dNTP 0.5 μL, and ddH2O is used to make up to 25 μL.

[0030] The PCR amplification reaction procedure in the S7 step is as follows: the first cycle is pre-denaturation at 94°C for 4 minutes; then denaturation at 94°C for 45 seconds, annealing at 54°C for 45 seconds, and extension at 54°C for 45 seconds, for a total of 35 cycles; and finally extension at 72°C for 10 minutes.

[0031] The voltage of electrophoresis in the step S8 is 4-5 V / cm.

[0032] See also Figures 1 to 8 The present invention makes full use of the characteristics of mature basidiospore ejection, directly fixes the diseased tissue on the culture dish cover for isolation and culture, greatly reduces the contamination of foreign bacteria, improves the culture efficiency, and ensures that it is the pathogen of tea cake disease. The rDNA-ITS region of the pathogen is amplified and confirmed by PCR using the universal primers ITS1 and ITS4 of the genomic transcription spacer region, which enhances the selectivity of the target strain and can quickly identify the pathogen. The specific method steps and principles are as follows: First, disease samples were collected. Diseased leaves were collected in tea gardens, placed in collection bags, stored at low temperatures, and brought back to the laboratory. Diseased leaves were then pretreated. Among the diseased leaves, some young, vigorous, and early diseased leaves that were not contaminated by other fungi were selected and rinsed with running tap water for 5-10 minutes. Fresh diseased spot tissue of 5mm×5mm was cut at the junction of diseased and healthy leaves with a sterile scalpel on a sterile operating table. After disinfection and rinsing in a sterile culture dish, 75% alcohol was used for disinfection 3 times, each time for 30 seconds. After disinfection, the leaves were rinsed with sterile water 3 times, each time for 1 minute.

[0033] Then, the pathogens were isolated. The washed leaves were glued to the PDA plate cover with the back facing down. The leaves were placed in an incubator at 25 degrees Celsius and placed in an upright position under light to allow the spores to fall freely. The incubator temperature was set to 25°C because basidiospores can germinate in the range of 15-32°C, the optimum temperature is 25°C, and germination stops at above 35°C. Then, the mycelium was picked for purification and culture. The diseased tissue was directly fixed on the culture dish cover for isolation and culture using the characteristics of mature basidiospore ejection. After about 20 days of culture, when the mycelium was white velvety and slightly shiny, the tip of the mycelium was gently picked at the edge of the colony and moved to the fresh tea juice PDA plate for purification and culture.

[0034] In the pathogen verification stage, the purified tea cake disease pathogen is punched and inoculated on the young leaves of the tea tree. The tea cake disease symptoms appear and a white fruiting layer is produced. Then the tea cake disease pathogen strain is re-isolated from the diseased detached leaf lesions. In the next pathogen DNA extraction stage, the fungal genomic DNA rapid extraction kit is used to extract DNA. The fungal genomic DNA rapid extraction kit of Beijing Adlai Biotechnology Co., Ltd. can be used. The precipitation is added with a suitable volume of sterile double distilled water or TE buffer, and stored at 4°C for use or stored at -20°C for long-term storage.

[0035] The PCR amplification stage uses the universal primers ITS1 and ITS4 of the genomic transcribed spacer region to PCR amplify the rDNA-ITS region of the pathogen (ITS1Forward: TCCGTAGGTGAACCTGCG; ITS4Reverse: TCCTCCGCTTATTGATAT). The reaction system for PCR identification is 25μL: PCR Buffer 2.5μl, 1μL of each primer, 1μL of DNA template, 0.4μL of TaqDNA polymerase, 0.5μL of dNTP, and ddH2O to make up to 25μL. The reaction program is the first cycle of 94℃ pre-denaturation for 4 minutes; then 94℃ denaturation for 45 seconds, 54℃ annealing for 45 seconds, 54℃ extension for 45 seconds, a total of 35 cycles; and finally 72℃ extension for 10 minutes.

[0036] The PCR identification stage is to electrophoresing the PCR amplification products on 1.0% agarose gel in 0.5×TBE buffer at a voltage of 4-5V / cm. After EB staining, the gel imager is placed to obtain the separation of PCR products. If it is the pathogen of tea cake disease, there will be a specific amplification band near 540bp.

[0037] Using the separation method of the present invention, among the 60 diseased tissues tested, 31 tea cake disease pathogen colonies were grown by spore bullet method, and no other bacteria and fungi were found. However, only 3 colonies were obtained by conventional method, and most of them were contaminated by other bacteria. See attached Figure 6 -Attached Figure 8 .

[0038] Compared with the prior art, the present invention uses the spore ejection method to separate pathogens, the contamination efficiency of endophytes is extremely low, and the separation and purification efficiency is high. Figure 5 Table. The pathogen of tea cake disease is a living parasite, and the diseased leaves collected in the field need to be separated immediately. At the same time, the tea trees suffering from tea cake disease have low resistance and are often accompanied by the parasitism of other pathogens. In addition, the pathogen of tea cake disease grows abnormally slowly. Most of the conventional tissue separation methods separate other pathogens that are highly saprophytic and grow vigorously. The present invention utilizes the characteristics of mature basidiospore ejection to directly fix the diseased tissue on the cover of the culture dish for separation and culture. The use of the present invention can accurately obtain the target pathogen, laying a solid foundation for subsequent research.

[0039] The above content is a further detailed description of the present invention in combination with specific implementation methods. It cannot be considered that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, simple modifications and substitutions made without departing from the concept of the present invention should be deemed to belong to the scope of protection of the present invention.

Claims

1. A method for separating, purifying and identifying the pathogenic bacteria of tea cake disease, characterized in that The following steps are involved: S1. Disease sample collection: Collect diseased leaves from the tea garden, put them into a collection bag, store them at low temperature, and bring them back to the laboratory; S2. Pretreatment of diseased leaves: Select some relatively tender, vigorous diseased leaves that are not contaminated by other fungi and are in the early stage of the disease from the picked diseased leaves, rinse them with running tap water for 5-10 minutes, cut 5mm×5mm fresh diseased spot tissue at the junction of diseased and healthy leaves with a sterile scalpel on a sterile operating table, disinfect them in a sterile culture dish and rinse them; S3. Isolation of pathogens: Stick the rinsed leaves with the back side facing down on the PDA plate cover, and incubate them in an incubator at 25 degrees Celsius with light, allowing the spores to fall freely; S4. Pick hyphae for purification and culture: Using the characteristics of mature basidiospore ejection, directly fix the diseased tissue on the cover of the culture dish for separation and culture. After culturing for about 20 days, when the hyphae are white velvety and slightly shiny, gently pick the hyphae tip at the edge of the colony and move it to the fresh tea juice PDA plate for purification and culture; S5. Pathogen verification: Punch the purified tea cake disease pathogen obtained, inoculate it on the young leaves of tea plants, and culture it under heat and moisture preservation until the symptoms of tea cake disease appear and a white hymenium is produced, and then re-isolate the tea cake disease pathogen strain from the diseased detached leaf spots; S6. Pathogen DNA extraction: Use the Fungal Genomic DNA Rapid Extraction Kit to extract DNA, add an appropriate volume of sterile double distilled water or TE buffer to the precipitate, and store at 4°C for later use or at -20°C for long-term storage; S7, PCR amplification: PCR amplification of the pathogen rDNA-ITS region was performed using universal primers ITS1 and ITS4 of the genomic transcribed spacer region; S8. PCR identification: The PCR amplification product was electrophoresed on a 1.0% agarose gel in 0.5×TBE buffer. After EB staining, it was placed on a gel imager to obtain the separation of the PCR products. If it is the pathogen of tea cake disease, there will be a specific amplification band near 540 bp.

2. The method for separating, purifying and identifying the pathogen of tea cake disease according to claim 1, characterized in that: In the step S2, the sterile culture dish is disinfected with 75% alcohol for 3 times, each time for 30 seconds; during rinsing after disinfection, it is rinsed with sterile water for 3 times after each alcohol disinfection, each time for 1 minute.

3. The method for separating, purifying and identifying the pathogen of tea cake disease according to claim 1, characterized in that: In the step S4, the temperature of the incubator is set to 15-32°C.

4. A method for separating, purifying and identifying the pathogen of tea cake disease according to claim 3, characterized in that: The optimum temperature of the incubator in step S4 is 25°C.

5. A method for separating, purifying and identifying the pathogen of tea cake disease according to claim 1, characterized in that: The reaction system for PCR amplification in step S7 is 25 μL: PCR Buffer 2.5 μL, primers 1 μL each, DNA template 1 μL, Taq DNA polymerase 0.4 μL, dNTP 0.5 μL, and ddH2O is used to make up to 25 μL.

6. A method for separating, purifying and identifying the pathogen of tea cake disease according to claim 1, characterized in that: The PCR amplification reaction procedure in the S7 step is as follows: the first cycle is pre-denaturation at 94°C for 4 minutes; then denaturation at 94°C for 45 seconds, annealing at 54°C for 45 seconds, and extension at 54°C for 45 seconds, for a total of 35 cycles; and finally extension at 72°C for 10 minutes.

7. A method for separating, purifying and identifying the pathogen of tea cake disease according to claim 1, characterized in that: The voltage of electrophoresis in the step S8 is 4-5 V / cm.

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