Some standard content:
GB18133-2000
With the development of my country's economy, international trade and technological cooperation, it is necessary to improve the quality of my country's potato seed potatoes as soon as possible and make them in line with international standards. It is necessary to formulate my country's potato virus-free seed potato quality standards. Appendix A, Appendix B, Appendix C, Appendix D and Appendix E in this standard are all appendices to the standard. This standard is proposed and managed by the Ministry of Agriculture of the People's Republic of China. The drafting units of this standard are: Potato Research Institute of Heilongjiang Academy of Agricultural Sciences, Grain and Product Quality Supervision and Inspection and Testing Center of the Ministry of Agriculture (Harbin), and Biotechnology Research Center of Heilongjiang Academy of Agricultural Sciences. The main drafters of this standard are: Cui Rongchang, Li Zhifang, Wu Guolin, Wang Lekai, Lei Bojun, and He Shitao. 331
1 Scope
National Standard of the People's Republic of China
Certified seed potatoes
GB18133—2000
This standard specifies the quality indicators and test methods for virus-free potato seedlings and virus-free potato seed potatoes of all levels. This standard applies to the quality identification of virus-free potato seedlings and virus-free seed potatoes during production and sales. 2 Definitions
This standard uses the following definitions.
2.1 Virus-free seedlings
Regenerated test tube seedlings obtained by stem tip tissue culture technology are confirmed to be virus-free only after being tested and confirmed to be free of viruses such as potato virus X (PVX), potato virus Y (PVY), potato virus S (PVS), potato leaf roll virus (PLRV) and potato spindle tuber viroid (PSTVd).
2.2 Virus-free seed potatoes
Produced by a seed potato production system that starts with breeding virus-free seedlings and increases the number of seed potatoes through successive generations of breeding. Virus-free seed potatoes are divided into two categories: basic seed potatoes and qualified seed potatoes. Basic seed potatoes refer to the original seeds and original seeds used to produce qualified seed potatoes; qualified seed potatoes refer to the seed potatoes used to produce commercial potatoes. 2.2.1 Basic seed potatoes: divided into three levels.
2.2.1.1 Pre-elite
Microtubers (Microtuber) produced in containers using virus-free seedlings and seed potatoes or minitubers (Minituber) that meet quality standards produced under insect-proof nets and greenhouse conditions.
2.2.1.2 Elite 1
Seed potatoes that meet quality standards are produced under good isolation conditions using original seeds as seed potatoes. 2.2.1.3 Elite I
The seed potatoes that meet the quality standards are produced under good isolation conditions using the first-level seed as seed potatoes. 2.2.2 Qualified seed potatoes: divided into two levels.
2.2.2.1 Certified grade 1 Seed potatoes that meet the quality standards are produced under isolation conditions using the second-level seed as seed potatoes. 2.2.2.2 Certified grade 1 Seed potatoes that meet the quality standards are produced under isolation conditions using the first-level seed potatoes as seed potatoes. 2.3 Permissible rate of virus-infected plants
The permissible rate of virus-infected plants in the virus-free seed potato breeding field. 2.4 Permissible rate of bacterial diseased plants
The permissible rate of bacterial diseased plants in the virus-free seed potato breeding field. 2.5 Permissible rate of mixed plants
The rate of plants of other potato varieties mixed in the virus-free seed potato breeding field. Approved by the State Administration of Quality and Technical Supervision on June 13, 2000 332
Implemented on October 1, 2000
2.6 Defective potatoes
GB18133-2000
Potatoes with deformity, secondary growth, cracking, insect damage, frostbite, black heart and mechanical damage. 3 Diseases to be controlled and eliminated
3. 1 Diseases to be controlled
3.1.1 Potato virus disease: Plants with virus disease symptoms such as mosaic, leaf curling, stripe necrosis, etc. in the virus-free seed potato breeding field. 3. 1.2 Potato black leg disease [Erwinia var. atroseptica (Van Hall) Dye.J or Erwinia var. carotovora (JonesDye.)
3.1.3 Potato bacterial wilt (Pseudomonus solanacearum EFSmith). 3.2 Diseases to be eliminated
3.2.1 Potato spindle tuber viroid (PotaoSpindleTuberViroid.PSTVd). 3.2.2 Potato bell ring rot [Corynebacterium Sepedonicum （Sieck &.Kott.） Skapt & Burkh]. 3.2.3 Potato tumor [Synchytrium endobaiticum（Schilb,）Perc.1.4 Quality requirements
4.1 The disease index of plants in virus-free seed potato fields of various levels shall meet the requirements of Table 1. Table 1 Allowable rate of diseased plants in seed potatoes of various gradesFirst inspection
Diseases and mixed plants, %
Seed grade
Original seed
First-level original seed
Second-level original seed
First-level seed potato
Second-level seed potato
Virus black leg
<0. 250. 5 K0. 25
0.5/≤1.0
Second inspection
Diseases and mixed plants, %
The tuber quality indicators of second-level seed potatoes should meet the requirements of Table 2. Virus
K0. 25≤0.50.1
1.0≤2.040. 1
Tuber quality indicators of seed potatoes
Tuber diseases and defects
Ring rot
Wet rot and rot
Dry rot
Sore rot, black rot and late blight:
Mild symptoms (1% ~ 5% tuber surface has spots) Moderate symptoms (5% ~10% tuber surface has disease spots) defective potatoes (except frostbite)
Third inspection
Disease and mixed plants, %
Allowance rate, %
5 Inspection method
5.1 Inspection of virus-free seedlings
GB18133-2000
5.1.1 Inspection after virus-free
After a batch of tissue culture regeneration seedlings of a certain variety is obtained, the virus and viroid status of each plant should be identified. Only tissue culture seedlings without potato X, S, Y viruses, potato leaf roll virus (PLRV) and viroids can be confirmed as virus-free seedlings. The detection method should meet the requirements of Appendix A and Appendix B.
5.1.2 Inspection before propagation
Before propagation, virus-free seedlings must be tested again to confirm that they are free of potato viruses X, S, Y, potato leaf roll virus (PLRV) and viroids before they can be used for propagation. The detection method should comply with the requirements of Appendix A and Appendix B. 5.2 Inspection of virus-free seed potatoes
5.2.1 Visual inspection of plant quality shall be carried out in the field where virus-free seed potatoes are planted. The detection method should comply with the requirements of Appendix C. If further confirmation is required, the method listed in Appendix D shall be used for inspection. 5.2.2 Number of inspections
After an overall observation of the plants growing in the field, random sampling shall be carried out for inspection according to the method in Table 3 and recorded in Table 4. Table 3 Number of inspection points and number of plants in fields of different sizes Area, hm2
Number of inspection points and number of inspection plants at each point
Random sampling inspection of 2 points, 100 plants at each point
Random sampling inspection of 5 points, 100 plants at each point
Random sampling inspection of 10 points, 100 plants at each point
Random sampling inspection of 10 points, 100 plants at each point For areas exceeding 5hm2, another inspection area shall be designated according to the different areas specified in this standard. Inspection points for the area, number of plants to be inspected in Table 4 Inspection records
Field inspection records for virus-free potato seeds for single seed propagation unit:
5.2.3 Inspection of original seed and original seed
Seed potato level:
Area:
Number of various diseased plants
Stripe and spot lesions
Dead diseased plants
Inspection date:
Variety:
Inspector
(Signature)
GB 18133--2000
Three inspections should be conducted during the growth period. The first inspection is at the budding stage of the plant, the second at the flowering stage, and the third two weeks before the withering and yellowing stage. The inspection shall be conducted by the seed propagation unit, and the inspection records shall be made and reported to the inspection department for filing. When a review is required, the inspection department shall send personnel to conduct a review inspection. 5.2.4 Inspection of first-level and second-level seed potatoes Two field inspections shall be conducted during the growth period. The inspection time is the same as the first and second inspection time of the original seed and original seed. 5.3 Tuber quality inspection of seed potatoes
When selling, randomly select tuber samples of 1% of the total amount of seed potatoes for tuber quality inspection. 5.4 Inspection of the allowable rate of mixed tubers of seed potatoes
When selling, the allowable rate of mixed tubers of seed potatoes shall be inspected according to the characteristics of tuber shape, skin color, flesh color, eye depth, etc. If further confirmation is required, the mixed tuber ratio of seed potatoes shall be inspected according to the polyacrylamide gel electrophoresis method of potato water-soluble protein in Appendix E. 6 Judgment rules
6.1 The grading of virus-free seed potatoes shall be based on the grade of seed potatoes sown in the virus-free seed mushroom breeding field, the rate of diseased plants and the rate of mixed plants as the grading standards. 6.2 For the three quality indicators of the rate of virus-infected plants, the rate of plants with black shank and back blight, and the rate of mixed plants of virus-free seed potatoes of each level, if any one of them does not meet the original level of plasmolysis standard but is higher than the next level of quality standard, the judgment result shall be graded down by one level. 6.3 After passing the inspection and grading, the potato virus-free seed potato quality inspection certificate shall be issued (see Table 5). Table 5 Quality Inspection Certificate for Virus-free Potato Seed Potato Quality Inspection Certificate for Virus-free Potato Seed Potato Year
Seed potato production unit:
Seed potato grade:
Variety:
Weight:
Number:
The above-mentioned seeds have passed the inspection according to the national standard of the People's Republic of China (GB 18133--2000 "Potato Virus-free Seed Potatoes") and this certificate is hereby issued. Inspector:
Inspection authority:
7 Packaging and labeling
7.1 Packaging
7.1.1 Packed in clean gunny bags.
(Signature)
(Seal)
7.1.2 Each gunny bag contains 80kg of virus-free seed potatoes. 7.2 Labels for virus-free potato seed potatoes
7.2.1 The label material should be made of soft white polyethylene film with a thickness of 0.10~0.12mm. It should meet the specifications of Figure 1. 7.2.2 The items on the front of the label should be printed in black No. 5 Song font, and the back of the label should be blank. In the item column on the front of the label, fill in the items to be recorded with a black marker or blue ballpoint pen. 7.2.3 Put a label in the sack and tie a label on the upper part of the sack. 335
8 Transportation
Potato virus-free seed potatoes
Certificate of conformity
Production unit
GB18133-2000
Figure 1 Labels tied to the packaging bag of virus-free potato seed potatoes 8.1 Seed potatoes of different varieties and grades should be strictly prevented from mixing when transported together. 8.2 Prevent mechanical damage, rain, heat and frost. 336
GB18133--2000
Appendix A
(Standard Appendix)
Enzyme-Linked Immunosorbent Assay
(Enzyme-Linked Immunosorbent Assay) Double Antibody Sandwich Method is used to detect whether potato virus-free seedlings carry potato X, Y, S viruses and potato leaf roll virus (PLRV) and other major potato viruses. A1 Instruments and Equipment
A1.1 Step ethylene microtiter plates have two specifications of 40 holes and 96 holes, both of which can be used. A1.2 Micro-adjustable injector: three specifications of 2~10μL, 10~~50μL and 10~200μL are required, and plastic heads of corresponding specifications are attached.
A1.3 Refrigerator.
A1.4 Incubator: set the temperature to 37℃.
A1.5 Small mortar made of glass or white porcelain. A1.6 Enzyme-linked immunosorbent assay: detect the enzyme-labeled antibody labeled with horseradish peroxidase (HRP.) at a wavelength of 490nm, and the enzyme-labeled antibody labeled with alkaline phosphatase (AKP) at a wavelength of 405nm. A2 The reagents used are analytical grade, and the water used is distilled water. A2.1 Antibody immunoglobulin (r-globulin) and enzyme-labeled antibody (Conjugate): The immunoglobulin extracted from a potato virus antiserum is adjusted to a concentration of 1mg/mL as the antibody for coating the microtiter plate. The enzyme-labeled antibody of the immunoglobulin of a virus labeled with horseradish peroxidase is generally used at a concentration of more than 1:1000. Store at 4℃ for use. A2.2 Carbonate coating buffer, pH 9.6: 1.59 g sodium carbonate (NaCO3), 2.93 g sodium bicarbonate (NaHCO), add water to 1 L. A2.3 PBS-Tween-20 buffer, pH 7.4: 8 g sodium chloride (NaCl), 0.2 g potassium dihydrogen phosphate (KH,PO.), 2.2 g disodium hydrogen phosphate (NazHPO,7H,O) (or 2.9 g NazHPO,-12H2O), 0.2 g (potassium chloride) KC1, add water to 1 L, then add 0.5 mL Tween-20. For washing microtiter plates. A2.4 Sample buffer: Take 100 mL of PBS-Tween-20 buffer and add 2 g of polyvinyl pyrrolidone (PVP). A2.5 Substrate buffer: Take 25.7mL of 0.2mol/L NazHPO.12H2O solution and add 24.3mL of 0.1mol/L citric acid solution, add 50mL of water, pH 5.0 (prepare immediately before use). Add 40mg of phosphorophenylenediamine and 0.15mL of 30% hydrogen peroxide (H2Oz) before use, mix well, and place away from light. It should be a white or slightly yellow solution. A2.6 Stop solution: 0.2mol/L sulfuric acid solution. Add 9 parts of water to 1 volume of concentrated sulfuric acid. A3 Operation steps
A3.1 Coating microtiter plate: Dilute the immunoglobulin with coating buffer at 1:1000, and add 200μL of diluted immunoglobulin to each sample well of the microtiter plate using a microinjector. Incubate at 37℃ for 1h, or overnight at 4℃. A3.2 Washing the coated microtiter plate: Shake off the immunoglobulin diluent in the microtiter plate, and then tap the microtiter plate on a stack of absorbent paper to remove all residual solution. Fill the sample wells of the microtiter plate with washing buffer, leave for 3 minutes, shake off the washing buffer, and wash 3 times in total to remove all unabsorbed immunoglobulins. A3.3 Adding the sample to be tested
A3.3.1 Sampling: Under sterile conditions, cut a 2cm long stem segment from the test tube seedling, place it in a small mortar, put the sampled test tube seedling back into the test tube, and seal the tube. Number the samples so that they can be selected according to the test results. 337
GB18133—2000
A3.3.2 Add sample buffer to the small mortar. The amount of liquid added depends on the number of wells for each sample. For example, when each sample is ready to be loaded into one sample well, 0.4 mL of sample buffer can be added. After grinding, 200 uL of clear solution can be obtained, which is enough for one sample well. A3.3.3 Add test samples: Add 200 μL of extracted sample solution to the sample wells of the numbered and washed microtiter plates one by one according to the sample number. Two positive control wells, two negative control wells and two blank control wells can be set on each microtiter plate. A3.3.4 Incubate the microtiter plate with added samples at 37°C for 4 to 6 hours, or overnight at 4°C, and then wash the microtiter plate according to A3.2.
A3.3.5 Add enzyme-labeled antibody: Dilute the enzyme-labeled antibody with sample buffer at 1:1000, and add 200 uL of diluted enzyme-labeled antibody to each sample well.
A3.3.6 Wash the microtiter plate: Wash the microtiter plate according to A3.2 to remove the unbound enzyme-labeled antibody. A3.3.7 Add substrate: Add 100μL of substrate buffer to each sample well. This is because when using a domestic enzyme-labeled detector to measure optical density, if the amount of substrate is large, it often contaminates the detection lens, making the measured optical density value inaccurate; if an imported enzyme-labeled detector such as Bio-Rad550 is used, 200μL of substrate can be added. When it is observed that the colors of the positive control wells and the negative control wells can be clearly distinguished (the enzyme-labeled antibody labeled with horseradish peroxidase appears orange-red, and the antibody labeled with alkaline phosphatase appears bright yellow); or when the colors of some sample wells of the microtiter plate without control sample wells can be clearly distinguished, add 30μL of stop solution to each well. If 200μL of substrate buffer is added, add 50μL of stop solution (the antibody labeled with alkaline phosphatase generally does not need to be added with stop solution). A4 Result determination
A4.1 Visual observation: The depth of color is proportional to the relative concentration of the virus. White indicates a negative reaction, recorded as "one"; light orange indicates a positive reaction, recorded as "ten"; as the color gradually deepens, it is recorded as "ten ten" and "ten ten ten". A4.2 Use an enzyme-linked detector to measure the optical density value: if the optical density value of the sample well is greater than 2 times the optical density value of the negative control well, it is judged as a positive reaction (the optical density value of the negative control well should be ≤0.1). Appendix B
(Standard Appendix)
Reciprocating two-dimensional polyacrylamide gel electrophoresis (Two-dimensional polyacrylamide Gel Electrophoresis) This method is used to detect whether the virus-free seedlings are infected with potato spindle tuber virus. B1 Instruments and equipment
B1.1 Electrophoresis instrument.
B1.2 Electrophoresis tank.
B1.3 Centrifuge with a speed of more than 3000r/min. B1.4 Refrigerator.
B1.5 High-temperature water bath.
B1.6 Micro-adjustable sampler, with three specifications of 2~10μL, 10~~50μL, and 10~200μL, and with plastic heads of corresponding specifications. B1.7 Small mortar made of glass or white porcelain. B1.8 Small glass pipette with nipple.
B1.9 Small plastic plate.
B1.10 Toothpicks, filter paper, etc.
B2 Reagents
Analytical grade specifications, water is distilled water 338
GB18133-2000
B2.1 Nucleic acid extraction buffer: 0.53mol/L ammonia (NH,OH), 0.013mol/L sodium ethylenediaminetetraacetate [(Naz-EDTA), adjusted to pH7.o with tris(hydroxymethyl)nitromethane (Tris)], 4 mol/L lithium chloride (LiCl). B2.210X electrode buffer: 0.89 mol/L Tris, 0.89 mol/L boric acid, 2.5mmol/L Na2-EDTA, pH8.3. B2.3 Sample loading buffer: 60mL 1X electrode buffer plus 40mL glycerol, containing 0.25% xylene cyanol and 0.25% bromophenol blue. B2.4 Water-saturated phenol: an aqueous solution of about 80% phenol, containing 0.1% 8-hydroxyoxazoline. B2.5 30% acrylamide stock solution: 30g acrylamide, 0.75g methylenebisacrylamide, dilute to 100mL with water, filter, and store at 4°C.
B2.6 10% ammonium persulfate solution: 0.1g ammonium persulfate plus 1mL of water (prepare immediately before use). B2.7 Tetramethylethylenediamine (TEMED).
B2.8 4mol/L sodium acetate solution: 5.44g anhydrous sodium acetate dilute to 10mL. B2. 9 Urea: add 8 mol/L to the reverse electrophoresis gel, i.e. the denaturing gel. B2.10 Nucleic acid fixative: an aqueous solution containing 10% ethanol and 0.5% acetic acid. B2.11 0.2% silver nitrate solution.
B2.12 Nucleic acid developer: 0.375mol/L sodium hydroxide (NaOH), 2.3mmol/L sodium borohydride (NaBH), 0.4% formaldehyde (37%, W/V).
B2.13 Color enhancer: 70 mmol/L sodium carbonate (Na2COs) aqueous solution. B3 Nucleic acid extraction
B3.1 Under sterile conditions, cut a 2cm long stem segment from the test tube seedling and place it in a small mortar. Put the sampled test tube seedling back into the test tube, seal the tube mouth, and number it so that you can decide whether to choose it or not according to the test results. B3.2 Add 0.4mL nucleic acid extraction buffer and 0.6mL water-saturated phenol to the small mortar, grind it, and pour it into a small plastic centrifuge tube. B3.33000r/min (it can be centrifuged within the range of 3000~8000r/min, generally the higher the centrifugation speed, the better) Centrifuge for 15min. Use a small pipette with a nipple to suck the upper aqueous phase into another clean centrifuge tube. B3.4 Add 1mL of ethanol and 1 drop of 4mol/L sodium acetate to the small centrifuge tube, mix well and place in the refrigerator ice box for at least 30 minutes. B3.5 Centrifuge at 3000r/min for 15 minutes, pour off the ethanol, and gently rinse the tube wall twice with a small amount of ethanol: turn the centrifuge tube upside down and drain the remaining ethanol.
B3.6 Add 50μL of sample loading buffer to each centrifuge tube, mix well with a clean toothpick, and then use it for sample electrophoresis. B4 Electrophoresis
B4.1 Forward electrophoresis: Use 5% polyacrylamide gel and 1X electrode buffer to perform electrophoresis from negative electrode to positive electrode (the electrode of the upper electrophoresis tank is the negative electrode, and the lower electrophoresis tank is the positive electrode), with a current of 5mA per centimeter of gel width. The sample loading volume is 6μL. When the xylene cyanol tracer dye migrates to the middle of the gel plate (about 6 cm from the sample loading origin), fold the glass plate, cut a gel strip with a width of about 1 cm with a xylene cyanol band, and move it horizontally to the bottom edge of the glass plate. Install the glass plate and fill it with denaturing gel containing 8 mol/L adenosine; put the glass plate back into the electrophoresis tank, change the electrodes, and perform reverse electrophoresis from positive to negative. After about 20 minutes, when the xylene cyanol band has completely entered the denaturing gel, stop the electrophoresis. Disassemble the electrophoresis tank and remove the glass plate. B4.2 Heat the electrophoresis glass plate with denaturing gel to promote the denaturation of viroids. In an 80C water bath, heat the electrophoresis glass plate for 30 minutes, and then put the electrophoresis glass plate back into the electrophoresis tank.
B4.3 Reverse electrophoresis: It is electrophoresis from positive to negative. The electrode buffer and current are the same as those for forward electrophoresis. Electrophoresis for about 2 hours. When the xylene cyanol tracer dye band migrates to the top of the gel plate about 4 cm away from the electrophoresis origin, stop electrophoresis and take out the gel slice for silver staining. B5 Silver staining
B5.1 Nucleic acid fixation: Place the gel slice in a plastic dish containing 200 mL of nucleic acid fixative, gently shake for 10 minutes, and then pour out the fixative.
GB18133—2000
B5.2 Add 200 mL of 0.2% silver nitrate solution to the plastic dish, gently shake for 15 minutes, and then pour out the silver solution (reusable). B5.3 Rinse the gel plate with distilled water to remove the residual silver solution. Rinse four times, 200 mL of water each time, and rinse for 15 seconds each time. B5.4 Nucleic acid band color development: Add 200 mL of nucleic acid developer (prepared immediately before use), gently shake until the nucleic acid band is clearly visible, and then rinse with tap water to stop the development.
B5.5 Color enhancement: Add 200mL of color enhancement solution.
B5.6 Result determination: The nucleic acid band at the bottom quarter of the gel plate is the viroid nucleic acid band (i.e. the bottom nucleic acid band); the nucleic acid band above it is the host nucleic acid band. There is a gap between the host nucleic acid band and the viroid nucleic acid band, and the two can be clearly distinguished. Appendix C
(Standard Appendix)
Visual identification table of symptoms of major viral diseases, bacterial diseases and fungal diseases of potato According to the potato plant symptoms and tuber symptoms described in Table C1, visually inspect potato viroids, viruses and bacterial diseases. Table C1
Disease name
Potato spindle
Tuber viroid
Potato leaf roll
Potato mosaic
Potato ring rot
Potato black shank
Potato cream blight
Potato cancer
Plant symptoms
Tuber symptoms
! The infected tubers become longer and spindle-shaped, with more eye buds and bulging buds. The angle between the leaves and the main stem of the diseased plants is small and acute, and the leaves stand upright. The upper leaves become smaller. Sometimes the plants become dwarfed, and sometimes the tubers crack. The leaves curl, spoon-shaped or simple, with a brittle texture. The leaflets often have smaller tubers. Some varieties have brown chlorosis symptoms between the veins on the tuber cut surface, and some varieties have purple or yellow edges on the top of the leaves. Sometimes the plants are dwarfed and necrotic. The leaves are mottled or faded with yellow edges, and sometimes the mesophyll is convex! The tubers become smaller and wrinkled. Sometimes the back of the leaves have dark brown stripes and necrosis. In the late growth period, the leaves dry up and droop, but do not fall off. The leaves of one or more main stems of a clump of plants lose water and wilt, the leaf color is gray-green and the interveinal chlorosis symptoms appear. Soon the vascular bundles of the infected tubers soften and turn light yellow. When squeezed, the tissue collapses and becomes granular, and milky yellow bacterial pus is discharged. The surface leaf margins dry up and turn brown, and finally turn yellow and die. The dead leaves do not die! The vascular bundle part of the skin is separated from the potato flesh, and the potato skin has a reddish-brown net that falls off.
The diseased plants are short, with green leaves, rolled-up leaf margins, hard texture, and multiple lines.
The navel of the infected tuber is yellow and sunken, extending to the pith to form an angle between the leaves and the main stem. The base of the stem is dark brown and easy to pull out of the soil. In severe cases, the tuber rots inside.
The leaves of the diseased plants are gray-green, with rapid wilt, and the vascular bundle color is the color of the vascular bundle of the infected tuber. After cutting, the outer skin of the diseased part is milky white, and the stem The cross section is milky white, with sticky bacterial fluid overflowing. In severe cases, the tissues adjacent to the vascular bundle rot, often overflowing from the tubers. Generally, the plant grows normally; sometimes green fleshy tumors grow at the base of the stem in contact with the soil, which later turn brown and finally fall off. Bacterial pus flows out of the eyes of the grass. This disease occurs in the underground part of the plant, but the roots are not affected. Tumors can form on underground and aboveground buds, branches and tubers. The typical tumor is a rough, tender and fleshy sphere,It can grow into a large mass of cell proliferation tissue. Its color is similar to that of tubers and creeping branches. If exposed to the ground, it is green and black when aged. The symptoms on the tubers are very similar to those of cauliflower
GB 18133-2000
Appendix D
(Standard Appendix)
Method for identification of potato ring rot
Use Gram staining and eggplant inoculation identification method to identify whether potato virus-free seedlings and virus-free seed potatoes are infected with potato ring rot. When the Gram staining is positive, the eggplant inoculation identification method is used for identification, and a positive result is also produced, it can be confirmed that it is potato ring rot pathogen.
D1 Gram staining (Gram Stain)
D1.1 Test equipment
D1.1.1 Microscope.
D1.1.2 Slide.
D1.1.3 Alcohol lamp.
D1.2 Reagents
The reagents used are analytical grade, and the water used is distilled water, and some are sterile water. D1.2.1 Gentian violet staining solution: add 2.5g gentian violet to 1L of water. D1.2.2 Sodium bicarbonate solution: add 12.5g sodium bicarbonate (NaHCO) to 1L of water. D7.2.3 Iodine mordant solution: dissolve 2g iodine in 10mL 1mol/L sodium hydroxide (NaOH) solution, and add water to make it 100mL. D1.2.4 Decolorizer: add 75mL 95% ethanol to 25mL acetone. D1.2.5 Basic fuchsin counterstaining solution: take 100mL basic fuchsin 95% ethanol saturated solution, add water to 1L. D1.3 Preparation of smears
All test tools should be sterilized by wiping with 70% alcohol. D1.3.1 To identify plants, cut off the stem 2 cm above the ground surface, squeeze out the juice from the incision with tweezers, drop 1 drop on a glass slide, air-dry, and bake with an alcohol lamp 2 to 3 times to fix.
Another method is to cut off a 0.5 cm thick stem slice from one end of the incision, grind it in a small mortar, absorb 1 drop of juice and drop it on a glass slide, air-dry, and bake with a flame 2 to 3 times to fix.
D1.3.2 To identify tubers: Cut the tubers open. If there is pus or exudate in the vascular bundle, squeeze it with tweezers, drop 1 drop on a glass slide, add 1 drop of sterile water to dilute, air-dry, and bake with a flame 2 to 3 times to fix. If there is no exudate, use tweezers to take out some broken tissues near the vascular bundle and place them on a glass slide, add a drop of sterile water, and remove the broken tissues. After air-drying, bake with a flame 2 to 3 times to fix. D1.4 Smear staining
D1.4.1 Drop 1 drop of gentian violet and sodium bicarbonate equal volume mixture (prepared immediately before use) on the smear and stain for 20 seconds. D1.4.2 Drop 1 drop of iodine mordant solution and stain for 20 seconds, then wash with water. D1.4.3 Drop 1 drop of ethanol and acetone decolorizing solution, decolorize for 5~10 seconds, then wash with water. D1.4.4 Drop 1 drop of alkaline fuchsin solution and re-stain for 2~3 seconds, then wash with water and air dry. D1.5 Microscopic examination and result judgment
Use a microscope with a magnification of 1000~1500 times to examine. If a single bluish-purple bacterium or a cluster of two, three or four bacteria is seen, it is a ring rot bacteria and is judged as a positive reaction; if it is stained pink, it is not a ring rot bacteria and is judged as a negative reaction. D2 Eggplant inoculation identification method
D2.1 Host plant preparation
The Black Beauty variety is usually used. First, grow seedlings in a large flower box, and transplant them into a flower box filled with nutrient soil after germination, with one plant per pot. When the eggplant grows to 2~~3 weeks and the third true leaf appears, it can be used for inoculation identification. 341
D2.2 Inoculum preparation
GB 18133-—2000
Prepare the inoculum according to D1.3.1 and D1.3.2, and dilute it with an appropriate amount of sterile water. D2.3 Inoculation method
Use a 1mL BCG syringe to suck in the prepared inoculum, and use a No. 4 needle to perform acupuncture inoculation on the stem between the true leaf and cotyledon of the eggplant seedling, and acupuncture three positions for each eggplant seedling. The grafted eggplant seedlings were placed in a greenhouse at 20-25℃, with relative humidity above 70% and daily light for 12 hours.
D2.4 Symptoms and results judgment
One week after inoculation, water ulcer symptoms appeared on the edge of the first true leaf. After 12 days, the symptoms developed into water loss and wilting of the leaf edge or between the veins, chlorosis, and necrosis of the diseased part after 22 days, and the leaves grew deformed. If the eggplants produce the above symptoms after inoculation, it means that the inoculum contains potato ring rot bacteria, and the sample prepared from the inoculum is a positive reaction; if there are no symptoms, it is a negative reaction, which proves that the inoculum does not contain ring rot bacteria. Appendix E
(Standard Appendix)
Potato tuber water-soluble protein polyacrylamide gel electrophoresis identification method The electrophoresis spectrum of potato tuber water-soluble protein is used to identify the variety purity of tubers. E1 Instruments and equipment
E1.1 Electrophoresis instrument.
E1.2 Electrophoresis tank.
E1.3 Centrifuge with a speed of more than 3000r/min. E1.4 Refrigerator.
E1.5 Micro-adjustable injector, 2~10μL and 10~~50μL are required, and plastic heads of corresponding specifications are attached. E1.6 Small mortar made of glass or white porcelain. E1.7 Small glass pipette with nipple.
E1.8 Small plastic plate.
E2 Reagents
are analytical grade specifications, and the water used is distilled water. E2.1 Sodium sulfite solution: 1g sodium sulfite (Na2SO3) plus 0.75g sodium pyrosulfite (Na2S,Os) plus 20mL water. E2.2 Loading buffer: 5mg bromophenol blue plus 50mL water, keep the supernatant after low-speed centrifugation, and then add 25g sucrose. E2.330% acrylamide stock solution: 30g acrylamide, 1.5g methylbisacrylamide, dilute to 100mL with water, filter, and store at 4℃.
E2.410% ammonium persulfate solution: 0.1g ammonium persulfate plus 1mL water, prepare before use. E2.5 Tetramethylethylenediamine (TEMED).
E2.68X electrode buffer stock solution: 1mol/L tris (hydroxymethylaminomethane), 0.15mol/L boric acid, pH8.9. When using, add 7 parts of water to 1 part of the stock solution, which is 0.125mol/LTris, 0.02mol/L boric acid, pH8.9 electrode buffer. E2.70.025% Coomassie Brilliant Blue R250 staining solution: The preparation method is 160mL water, 40mL methanol, 14mL acetic acid, then add 12g trichloroacetic acid, and then add 5mL 1% Coomassie Brilliant Blue R-250 anhydrous ethanol solution, mix well. E2.8 Decolorizing solution: Prepare according to the ratio of 140mL water, 60mL methanol and 10mL acetic acid. E3 Extraction of water-soluble protein from tubers
E3.1 Take fresh or stored tubers, sample with a punch, remove the skin, weigh 5g of potato flesh and put it in a small mortar, add 0.5mL sulfurous acid 3422 Identify tubers: Cut the tubers. If there is pus or exudate in the vascular bundle, squeeze it with tweezers, drop 1 drop on the slide, add 1 drop of sterile water to dilute, air-dry, and fix it with flame baking 2-3 times. If there is no exudate, use tweezers to take out some broken tissues near the vascular bundle and place them on the slide, add drops of sterile water, and remove the broken tissues. After air-drying, fix it with flame baking 2-3 times. D1.4 Smear staining
D1.4.1 Drop 1 drop of gentian violet and sodium bicarbonate equal volume mixture (prepared immediately before use) on the smear and stain for 20 seconds. D1.4.2 Drop 1 drop of iodine mordant solution for mordant staining for 20 seconds, and wash with water. D1.4.3 Drop 1 drop of ethanol, acetone decolorizing solution, decolorize for 5-10 seconds, and wash with water. D1.4.4 Drop 1 drop of alkaline fuchsin solution for re-staining for 2-3 seconds, wash with water, and air-dry. D1.5 Microscopic examination and result determination
Use a microscope at 1000-1500 times to examine. If a single bluish-purple bacterium or a cluster of two, three or four bacteria is seen, it is a ring rot bacterium and is determined as a positive reaction; if it is stained pink, it is not a ring rot bacterium and is determined as a negative reaction. D2 Eggplant inoculation identification method
D2.1 Host plant preparation
The "Black Beauty" variety is generally used. First, grow seedlings in a large flower box, and after germination, transplant them into a flower box filled with nutrient soil, one plant per pot. When the eggplant grows to 2~~3 weeks and the third true leaf appears, it can be used for inoculation identification. 341
D2.2 Inoculum preparation
GB 18133-—2000
Prepare the inoculum according to the methods of D1.3.1 and D1.3.2, and dilute with an appropriate amount of sterile water. D2.3 Inoculation method
Use a 1mL BCG syringe to suck the prepared inoculum, and use a No. 4 needle to make a needle inoculation in the stem between the true leaves and cotyledons of the eggplant seedlings. Each eggplant seedling is punctured at three locations. The seeded eggplant seedlings are placed in a greenhouse at 20-25℃, with a relative humidity of more than 70% and a daily light of 12 hours.
D2.4 Symptom manifestation and result judgment
One week after inoculation, the first true leaf edge shows water ulcer symptoms. After 12 days, the symptoms develop into water loss and wilting of the leaf edge or between the veins, and chlorosis. After 22 days, the diseased part necrose and the leaves grow deformed. If the eggplant produces the above symptoms after inoculation, it means that the inoculum contains potato ring rot bacteria, and the sample prepared for the inoculum is a positive reaction; if there are no symptoms, it is a negative reaction, which proves that the inoculum does not contain ring rot bacteria. Appendix E
(Standard Appendix)
Potato tuber water-soluble protein polyacrylamide gel electrophoresis identification method The electrophoresis spectrum of potato tuber water-soluble protein is used to identify the variety purity of tubers. E1 Instruments and equipment
E1.1 Electrophoresis instrument.
E1.2 Electrophoresis tank.
E1.3 Centrifuge with a speed of more than 3000r/min. E1.4 Refrigerator. bZxz.net
E1.5 Micro-adjustable injector, 2~10μL and 10~~50μL are required, and plastic heads of corresponding specifications are attached. E1.6 Small mortar made of glass or white porcelain. E1.7 Small glass pipette with nipple.
E1.8 Small plastic plate.
E2 Reagents
It is of analytical grade specification, and the water is distilled water. E2.1 Sodium sulfite solution: 1g sodium sulfite (Na2SO3) plus 0.75g sodium pyrosulfite (Na2S,Os) plus 20mL water. E2.2 Sample loading buffer: 5mg bromophenol blue plus 50mL water, centrifuge at low speed, keep the supernatant, and then add 25g sucrose. E2.3 30% acrylamide stock solution: 30g acrylamide, 1.5g methylbisacrylamide, dilute to 100mL with water, filter, and store at 4℃.
E2.4 10% ammonium persulfate solution: 0.1g ammonium persulfate plus 1mL water, prepare before use. E2.5 Tetramethylethylenediamine (TEMED).
E2.6 8X electrode buffer stock solution: 1mol/L trishydroxymethylaminomethane (Tris), 0.15mol/L boric acid, pH8.9. When in use, add 7 parts of water to 1 part of the storage solution, which is 0.125mol/LTris, 0.02mol/L boric acid, pH8.9 electrode buffer. E2.7 0.025% Coomassie Brilliant Blue R250 staining solution: The preparation method is 160mL water, 40mL methanol, 14mL glacial acetic acid, then add 12g trichloroacetic acid, and then add 5mL 1% Coomassie Brilliant Blue R-250 anhydrous ethanol solution, mix well. E2.8 Decolorizing solution: Prepare according to the ratio of 140mL water, 60mL methanol and 10mL glacial acetic acid. E3 Tuber water-soluble protein extraction
E3.1 Take fresh or stored tubers, sample with a hole punch, remove the skin, weigh 5g of potato flesh and put it in a small mortar, add 0.5mL sulfurous acid 3422 Identify tubers: Cut the tubers. If there is pus or exudate in the vascular bundle, squeeze it with tweezers, drop 1 drop on the slide, add 1 drop of sterile water to dilute, air-dry, and fix it with flame baking 2-3 times. If there is no exudate, use tweezers to take out some broken tissues near the vascular bundle and place them on the slide, add drops of sterile water, and remove the broken tissues. After air-drying, fix it with flame baking 2-3 times. D1.4 Smear staining
D1.4.1 Drop 1 drop of gentian violet and sodium bicarbonate equal volume mixture (prepared immediately before use) on the smear and stain for 20 seconds. D1.4.2 Drop 1 drop of iodine mordant solution for mordant staining for 20 seconds, and wash with water. D1.4.3 Drop 1 drop of ethanol, acetone decolorizing solution, decolorize for 5-10 seconds, and wash with water. D1.4.4 Drop 1 drop of alkaline fuchsin solution for re-staining for 2-3 seconds, wash with water, and air-dry. D1.5 Microscopic examination and result determination
Use a microscope at 1000-1500 times to examine. If a single bluish-purple bacterium or a cluster of two, three or four bacteria is seen, it is a ring rot bacterium and is determined as a positive reaction; if it is stained pink, it is not a ring rot bacterium and is determined as a negative reaction. D2 Eggplant inoculation identification method
D2.1 Host plant preparation
The "Black Beauty" variety is generally used. First, grow seedlings in a large flower box, and after germination, transplant them into a flower box filled with nutrient soil, one plant per pot. When the eggplant grows to 2~~3 weeks and the third true leaf appears, it can be used for inoculation identification. 341
D2.2 Inoculum preparation
GB 18133-—2000
Prepare the inoculum according to the methods of D1.3.1 and D1.3.2, and dilute with an appropriate amount of sterile water. D2.3 Inoculation method
Use a 1mL BCG syringe to suck the prepared inoculum, and use a No. 4 needle to make a needle inoculation in the stem between the true leaves and cotyledons of the eggplant seedlings. Each eggplant seedling is punctured at three locations. The seeded eggplant seedlings are placed in a greenhouse at 20-25℃, with a relative humidity of more than 70% and a daily light of 12 hours.
D2.4 Symptom manifestation and result judgment
One week after inoculation, the first true leaf edge shows water ulcer symptoms. After 12 days, the symptoms develop into water loss and wilting of the leaf edge or between the veins, and chlorosis. After 22 days, the diseased part necrose and the leaves grow deformed. If the eggplant produces the above symptoms after inoculation, it means that the inoculum contains potato ring rot bacteria, and the sample prepared for the inoculum is a positive reaction; if there are no symptoms, it is a negative reaction, which proves that the inoculum does not contain ring rot bacteria. Appendix E
(Standard Appendix)
Potato tuber water-soluble protein polyacrylamide gel electrophoresis identification method The electrophoresis spectrum of potato tuber water-soluble protein is used to identify the variety purity of tubers. E1 Instruments and equipment
E1.1 Electrophoresis instrument.
E1.2 Electrophoresis tank.
E1.3 Centrifuge with a speed of more than 3000r/min. E1.4 Refrigerator.
E1.5 Micro-adjustable injector, 2~10μL and 10~~50μL are required, and plastic heads of corresponding specifications are attached. E1.6 Small mortar made of glass or white porcelain. E1.7 Small glass pipette with nipple.
E1.8 Small plastic plate.
E2 Reagents
It is of analytical grade specification, and the water is distilled water. E2.1 Sodium sulfite solution: 1g sodium sulfite (Na2SO3) plus 0.75g sodium pyrosulfite (Na2S,Os) plus 20mL water. E2.2 Sample loading buffer: 5mg bromophenol blue plus 50mL water, centrifuge at low speed, keep the supernatant, and then add 25g sucrose. E2.3 30% acrylamide stock solution: 30g acrylamide, 1.5g methylbisacrylamide, dilute to 100mL with water, filter, and store at 4℃.
E2.4 10% ammonium persulfate solution: 0.1g ammonium persulfate plus 1mL water, prepare before use. E2.5 Tetramethylethylenediamine (TEMED).
E2.6 8X electrode buffer stock solution: 1mol/L trishydroxymethylaminomethane (Tris), 0.15mol/L boric acid, pH8.9. When in use, add 7 parts of water to 1 part of the storage solution, which is 0.125mol/LTris, 0.02mol/L boric acid, pH8.9 electrode buffer. E2.7 0.025% Coomassie Brilliant Blue R250 staining solution: The preparation method is 160mL water, 40mL methanol, 14mL glacial acetic acid, then add 12g trichloroacetic acid, and then add 5mL 1% Coomassie Brilliant Blue R-250 anhydrous ethanol solution, mix well. E2.8 Decolorizing solution: Prepare according to the ratio of 140mL water, 60mL methanol and 10mL glacial acetic acid. E3 Tuber water-soluble protein extraction
E3.1 Take fresh or stored tubers, sample with a hole punch, remove the skin, weigh 5g of potato flesh and put it in a small mortar, add 0.5mL sulfurous acid 3420.25% Coomassie Brilliant Blue R250 staining solution: The preparation method is 160mL water, 40mL methanol, 14mL acetic acid, then add 12g trichloroacetic acid, then add 5mL 1% Coomassie Brilliant Blue R-250 anhydrous ethanol solution, mix well. E2.8 Decolorizing solution: Prepare according to the ratio of 140mL water, 60mL methanol and 10mL acetic acid. E3 Extraction of water-soluble protein from tubers
E3.1 Take fresh or stored tubers, sample with a cork punch, remove the skin, weigh 5g of potato flesh and put it in a small mortar, add 0.5mL sulfurous acid 3420.25% Coomassie Brilliant Blue R250 staining solution: The preparation method is 160mL water, 40mL methanol, 14mL acetic acid, then add 12g trichloroacetic acid, then add 5mL 1% Coomassie Brilliant Blue R-250 anhydrous ethanol solution, mix well. E2.8 Decolorizing solution: Prepare according to the ratio of 140mL water, 60mL methanol and 10mL acetic acid. E3 Extraction of water-soluble protein from tubers
E3.1 Take fresh or stored tubers, sample with a cork punch, remove the skin, weigh 5g of potato flesh and put it in a small mortar, add 0.5mL sulfurous acid 342
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