Method for rapidly separating single spores of camellia oleifera brown spot bacteria

Abstract

The invention belongs to the technical field of microorganisms, and particularly discloses a method for rapidly separating single spores of camellia oleifera brown spot bacteria. Comprising the following steps: collecting a sample; the method comprises the following steps: selecting scab tissues with obvious characteristics (symptoms) on a sample, slicing in a bare-handed manner, placing a slice material on a glass slide, and dropwise adding sterile water to prepare a bacterial spore-containing mixed solution; the method comprises the following steps: taking a glass sample application capillary tube with the specification of 0.3 * 100mm, firing the middle part of the capillary tube on the outer flame of an alcohol lamp, then lengthening the capillary tube until the capillary tube is broken within 3-5 seconds, cutting off the fired top end by using scissors, and inserting the unfired end into a gun head of a gene gun with the specification of 10-100 microliters; aligning a single spore in the mixed solution under a stereoscope, and touching and sucking the single spore; installing a gene gun on the head of the gene gun into which the monospores are sucked, and rapidly scribing and striking the sucked monospores into a PDA culture medium; culturing in a constant-temperature box; with the adoption of the technical scheme, the camellia oleifera brown spot fungus monospore can be directly obtained, the operation process is simple, the period is short, the success rate is high, and the method is applicable to treatment of large-batch samples.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for rapidly separating single spores of tea oil brown spot pathogen. Background Art

[0002] The pathogen of tea leaf spot is Pseudomonas oleifera ( Pestalotiopsis theae (Sawada) Steyaert, belongs to the class of fungi, the genus of Pseudomonas. This pathogen is the dominant species of brown spot disease of oil-tea camellia in China. This pathogen prefers high temperature and high humidity, and the conditions are suitable for its rapid spread. In some years, the incidence rate can reach 78%, causing leaf and fruit fall, causing huge losses to oil-tea.

[0003] Pseudosporium truncatum P. theae ) Overwinters in dead branches, fallen leaves and diseased debris with hyphae and conidia, and is the main source of infection for oil tea brown spot disease the following year. It often breaks out and spreads during the hot rainy season every year. In the early stage of the disease, the disease starts from the tip or edge of the leaf. The lesions are small, circular or nearly circular, with obvious diseased and healthy parts. Small dark brown dots can be seen in the center of the lesions. The lesions gradually expand and turn brown, causing brown spot symptoms. There may be ring patterns at the junction of the lesions and the diseased and healthy parts. The fruiting body in the center of the lesion is the conidiophore and conidia, which can be spread by the synergistic effect of wind and rain, causing the occurrence of oil tea brown spot disease. The small black dots on the lesions are the conidiophores of the pathogen, with a diameter of 120-180 microns. The dark black dots on the diseased part are the conidiophores of the pathogen. Conidiophores grow on it, which are colorless and filamentous. Conidia are spindle-shaped, with 4 septa and 5 cells. The 3 middle cells are yellow-brown or dark brown, the cells at both ends are small and colorless, and the top cell has 3-5 colorless stings. Initially buried in the palisade tissue under the host epidermis, it later breaks through the epidermis and is exposed. Conidiophores grow in clusters and are cylindrical. Conidia are spindle-shaped, with 4 septa in most cases, and are 20-30 microns × 6-8 microns in size. The top cell of the spore has 3 appendages, which are thick at the base and gradually become thinner upwards, and the top is knot-shaped and swollen.

[0004] The separation of the multi-hairy spore tissue of the brown spot of oil tea is easy to pollute. At present, there is no mature technology to produce a large amount of conidia on artificial culture medium. Therefore, the acquisition of the multi-hairy spore monospore strain of oil tea needs to be directly separated from the lesions of the leaf blades of the brown spot of oil tea in the wild. CN102140432A discloses a method for separating a single spore of fungi. The main steps are to insert a glass capillary into a Pasteur pipette from the tip, pick the fungal material to be separated with a dissecting needle, and directly face and approach the spore at the center of the capillary tube mouth, and transfer the spore to the top of the culture medium, and the single spore separation is completed. Its key step is to pick the fungal colony or lesion tissue of part to be separated with a dissecting needle, and for the lesion tissue, there may not be conidia on it, and directly take the lesion tissue to make a spore suspension, which has more impurities and is not easy to disperse the spore. In addition, the tool used includes a Pasteur pipette and a capillary, and a single spore is sucked into the capillary, and the rubber head is kept motionless, which requires a high level of technology and experience of the operator, and is not suitable for large-scale fungal single spore separation. CN108587929A discloses a method for rapidly separating single spores of rice blast fungi on rice blast lesions in the field, which mainly comprises coating rice leaves containing typical rice blast lesions on the surface of a water agar medium, culturing the water agar plate with rice blast fungi conidia, searching for single and germinated rice blast fungi conidia under a stereomicroscope, and then separating the single spores of rice blast fungi to obtain an agar block containing single spores of rice blast fungi. This method directly coats the surface of a water agar medium with lesions, which is not accurate in positioning conidia, has many other impurities, and requires cutting the agar block containing single spores, which has many operation steps and is relatively troublesome.

[0005] The disease mainly occurs on the leaves, and the fruiting bodies are relatively small. The needles used for traditional single spore separation have the following two disadvantages: (1) The area is too large, and in addition to the target single spores, it is easy to be contaminated with other bacteria, resulting in contamination and inaccuracy of the experimental results. (2) The leaves of fresh camellia are relatively soft. When using a needle to pick them up, due to the high hardness, it is very easy to pierce the target fruiting bodies, which can also easily cause inaccuracy in the experiment. This method can effectively circumvent the above two disadvantages. Therefore, in the study of the biological characteristics and pathogenic mechanism of the pathogen of Camellia oleifera brown spot disease, it is urgent to develop a single spore separation method that is fast, stable, efficient and suitable for large-scale sample separation. Summary of the invention

[0006] The main purpose of the present invention is to provide a method for quickly isolating single spores of tea oil brown spot pathogenic fungus, so as to reduce the contamination of miscellaneous bacteria and meet the needs of laboratory pathogen testing.

[0007] The present invention specifically provides the following technical solutions: A method for rapidly isolating single spores of tea oil brown spot pathogenic bacteria comprises the following steps: S1: Collection of oil tea samples showing brown spot disease; S2: Select the lesion tissue with obvious characteristics (symptoms) on the sample, slice it by hand, place the sliced ​​material on a glass slide, drop sterile water into it to prepare a mixture containing fungi and spores, observe it under a stereomicroscope, and confirm the presence of single conidia in the mixture; S3: Take a 0.3*100mm glass spotting capillary, burn the middle part of the capillary on the outer flame of an alcohol lamp, then stretch it within 3-5 seconds until it breaks, cut off the burned top with scissors, and insert the unburned end into the tip of a gene gun with a specification of 10-100 microliters; aim at a single spore in the mixed solution under a stereomicroscope, touch it, and inhale the single spore; S4: Install the gene gun tip for sucking single spores onto the gene gun, and quickly streak the sucked single spores onto the PDA medium; S5: The culture medium is sealed with a sealing film and placed in a constant temperature box for culture; S6: Observe and transfer the spores to new culture dishes when the spores germinate and grow into colonies.

[0008] Preferably, in step S1, when collecting tea oil samples, the samples are placed in envelope bags, and each sample is placed in an envelope bag separately.

[0009] Preferably, in steps S2 and S3, the stereoscopic mirror is a 10*10x stereoscopic mirror.

[0010] The advantages of the present invention compared with the prior art are: (1) The present invention proposes a new single spore isolation method for the pathogen of tea oil brown spot disease, filling the gap of the previous single spore isolation method for this pathogenic fungus. In addition, the present invention has a simple operation process, a short cycle, a high success rate, and is suitable for processing large quantities of samples.

[0011] (2) The present invention does not require the separate preparation of a conidia suspension, nor does it require dilution. Instead, the lesion site is sliced ​​directly by hand, and the cut material is placed in a drop of sterile water on a glass slide to form a spore-containing mixed solution. Conidia are directly obtained in a drop of water, which is convenient and fast, saves time, and improves efficiency.

[0012] (3) The present invention does not require the preparation of agar slides or the cutting of agar blocks. The present invention directly uses a gene gun tip (10-100 μl) to insert an elongated spotting capillary heated under an alcohol lamp to siphon single spores in the mixed solution, which has a more accurate separation efficiency and is not easy to be contaminated.

[0013] (4) The tools used in the present invention, such as the stereoscope, envelope, blade, glass slide, gene tube and gene gun, are all commonly used materials in laboratories. No special equipment needs to be purchased, and general laboratories can meet the requirements of the operation. In addition, the invention is simple to operate, and general experimental personnel can master it after 1-2 trainings. It is simple and easy to learn. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a sample of the air-dried tea leaf with brown spot disease (the black dots in the picture are the fruiting bodies of the pathogen); Figure 2 A spotting capillary (0.3*100mm) stretched by heating with alcohol is cut in the middle and inserted into the tip of the gene gun; Figure 3 To observe the individual spores in the mixed solution on the slide under a stereomicroscope, a pipette tip with a capillary tube was used to draw up the individual spores on-site; Figure 4 On-site photo of putting a yellow gun tip on the gene gun and streaking into the transparent PDA culture medium; Figure 5 Example 1: Single spore colony morphology after 5-6 days of culture in an incubator; Figure 6 The single spore colony morphology of other methods after 5-6 days of culture in an incubator, where A is the colony morphology of the spotting capillary (0.3*100mm) after siphoning without burning and stretching treatment; B is the colony morphology under the CN102140432A method. DETAILED DESCRIPTION Embodiment 1:

[0015] A method for rapidly isolating single conidia of tea oil brown spot pathogenic bacteria comprises the following steps: (1) Collect tea leaves with typical tea leaf brown spot disease in the wild ( Figure 1 ), the lesions should have obvious symptoms visible to the naked eye - small black spots, and then put them in a paper sampling envelope. Note that each leaf should be placed in an envelope separately and cannot be stacked. Take them back to the laboratory. If there is moisture on the surface of the leaves, they need to be dried naturally in a ventilated and dry place; (2) Select the part with obvious symptoms and slice it by hand. Use tweezers to take part of the lesion tissue cut into thin strips and place it on a glass slide with a drop of sterile water to form a mixture containing fungi and spores. Observe it under a 10*10x microscope to confirm the presence of single conidia in the mixture. Take a 0.3*100mm glass spot capillary (purchased from Nantong Surui Laboratory Equipment Co., Ltd.), burn the middle part of the capillary on the outer flame of an alcohol lamp, and then stretch it within 3-5 seconds until it breaks. Cut off the burned top with scissors, and insert the unburned end into the tip of a gene gun with a specification of 10-100 microliters ( Figure 2 ); aim at a single spore in the mixture under a stereomicroscope, touch it, and inhale the single spore ( Figure 3 ); (3) Then put on the gene gun and quickly draw a line of liquid onto the transparent PDA culture medium ( Figure 4PDA medium formula: 200 g potato, 20 g glucose, 15-20 g agar, 1000 ml distilled water, natural pH, at 121 o C for 20 minutes, pour into a plate and cool in a clean bench for later use.

[0016] (4) Seal with sealing film and place in a 25-degree incubator for incubation; (5) Observe every 4-8 hours. When the single spores germinate and grow into colonies, transfer them to a new culture dish. Figure 5 ).

[0017] like Figure 5 As shown, the single spore colony morphology after 5-6 days in the incubator using the method of the present invention, the present invention directly uses the gene gun tip (10-100 microliters) to insert the spotting capillary (0.3*100) that is heated, stretched, broken and the burned top is removed under an alcohol lamp to siphon the single spores in the mixed solution, which is easy to operate and has a more accurate separation efficiency. It can be seen from the figure that there are almost no foreign bacteria and it is not easy to be contaminated.

[0018] like Figure 6 As shown, the colonies after siphoning using the spotting capillary (0.3*100mm) without burning and stretching treatment and the colonies under the CN102140432A method separated many miscellaneous fungi of tea oil brown spot disease, and more steps of purification and separation are required to obtain single spores of tea oil brown spot disease fungi, which is time-consuming, labor-intensive, and increases the culture cost.

[0019] The above description of the present invention and its implementation methods is not restrictive. The drawings are also one of the implementation methods of the present invention, and the actual invention is not limited thereto. In short, if ordinary technicians in the field are inspired and design structural methods and implementation examples similar to the technical solution without creativity without departing from the purpose of the invention, they all fall within the protection scope of the present invention.

Claims

1. A method for rapidly isolating single spores of tea oil brown spot pathogen, characterized in that: The following steps are involved: S1: Collection of oil tea samples showing brown spot disease; S2: Select the lesion tissue with obvious characteristics on the sample, slice it by hand, place the sliced ​​material on a glass slide, drop sterile water into it to prepare a mixture containing bacteria and spores, observe it under a stereomicroscope, and confirm the presence of single conidia in the mixture; S3: Take a 0.3*100mm glass spotting capillary, burn the middle part of the capillary on the outer flame of an alcohol lamp, then stretch it within 3-5 seconds until it breaks, cut off the burned top with scissors, and insert the unburned end into the tip of a gene gun with a specification of 10-100 microliters; aim at a single spore in the mixed solution under a stereomicroscope, touch it, and inhale the single spore; S4: Install the gene gun tip for sucking single spores onto the gene gun, and quickly streak the sucked single spores onto the PDA medium; S5: The culture medium is sealed with a sealing film and placed in a constant temperature box for culture; S6: Observe and transfer the spores to new culture dishes when the spores germinate and grow into colonies.

2. The method according to claim 1, characterized in that: In step S1, when collecting tea oil samples, the samples are placed in envelope bags, and each sample is placed in an envelope bag separately.

3. The method according to claim 1, characterized in that In steps S2 and S3, the stereoscopic mirror is a 10*10x stereoscopic mirror.

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