GB 15569-1995 Quarantine regulations for transportation of agricultural plants
Some standard content:
National Standard of the People's Republic of China
Plant quarantine rules transportingof agricultural plant
Subject content and scope of applicationwwW.bzxz.Net
GB15569--1995
This standard specifies the quarantine procedures for the domestic transportation of agricultural plant seeds, seedlings and other propagation materials and quarantined machinery and plant products.
This standard applies to the transportation quarantine of various types of agricultural plant seeds, seedlings and other propagation materials and quarantined plants and plant products. 2 Terms
2.1 Transportation quarantine
refers to the quarantine inspection and visas conducted by professional agricultural plant quarantine personnel in accordance with plant quarantine regulations during the circulation (including consignment, mailing, self-transportation, carrying, sales, etc.) of various types of plant seeds, seedlings and other propagation materials and quarantined plants and plant products. 2.2 Plants and plant products subject to quarantine refer to plants and plant products subject to quarantine announced by the state and provinces (autonomous regions, municipalities directly under the central government). 2.3 Quarantine objects refer to agricultural plant quarantine objects announced by the state and supplementary quarantine objects announced by provinces (autonomous regions, municipalities directly under the central government). 2.4 Batch refers to all items subject to quarantine at the same time, at the same place of shipment and receipt, with the same product name (variety) and the same means of transport. 2.5 Piece refers to all items subject to quarantine in a package. 3 Transportation quarantine procedure See the figure below for the transportation quarantine procedure.
Approved by the State Bureau of Technical Supervision on June 2, 1995
Implemented on January 1, 1996
4 Preparatory work
On-site inspection
Sample sampling
No quarantine object
Visa approval
GB15569—1995
Transportation inspection line sequence
Special inspection
With quarantine Object
Processing format
Indoor inspection
Conventional inspection
Processing of unqualified
Prohibition of transportation or disposal
Other inspections
4.1 Accept and verify the application form for quarantine inspection of agricultural plants for transportation with a letter of introduction (or ID card), and submit the agricultural plant transportation quarantine requirements to be reviewed in Appendix B (supplementary) to the transfer-in place. Check the plant quarantine certificate during re-inspection. 4.2 Check the supply arrangement, transportation tools, packaging materials, etc. from the applicant, prepare quarantine tools, and determine the quarantine time, place and method. 5 On-site inspection
5.1 On-site inspection content
On-site inspection of agricultural plants, plant products and their packaging materials, transportation tools, stacking places, etc., to see if there are quarantine objects, pathogens and pests of various stages and their excrement, secretions, molting shells, holes and other traces. During the re-inspection, it shall be checked whether the transported agricultural plants and their products subject to inspection are consistent with the quarantine certificate.
5.2 Sampling
According to different agricultural plants and their products subject to inspection, different packages and different quantities, five diagonal sampling or stratified sampling shall be adopted. The samples shall be directly observed with the naked eye or a hand-held magnifying glass. 273
5.3 Inspection
GB15569—1995
Seeds shall be screened and inspected. For seedlings, scions, tubers, fruits, etc., the roots, branches, stems, inside and outside of the cortex, leaves, bud eyes and fruit surfaces, calyx pits, fruit stalks and other parts shall be carefully inspected for pests of various insect stages and holes, exits and insect worms; for disease symptoms, tumors, fungal worms, weed seeds, etc. See Table 1 for the specific number of random inspections.
Table 1 Standard table of number of on-site sampling
Seeds
Seedlings
Fruits, tubers
Chinese medicinal materials, tobacco
Percentage of sampling based on the total number of goods, %
≥4 000 kg 2~5
<4 000 kg 5~10
>10 000 plants 3~5
100~10 000 plants 6~10
0.2~~5.0
Note: ①) 100 kg of bulk seeds is one piece, and 100 seedlings is one piece. ②All items less than the minimum sampling number shall be inspected. Other categories can refer to the proportion in the table for random inspection.
6 Indoor inspection
6.1 Sampling
Minimum number of samples
100 pieces
100 plants
5 pieces or 100kg
6.1.1 If it is difficult to determine whether there are quarantine objects through on-site inspection, and indoor inspection is required or it is considered necessary to preserve samples, samples should be taken and brought back for indoor inspection or preservation in combination with on-site random inspection. 6.1.2 Sampling method
According to different agricultural plants and their products to be inspected, different packaging, different quantities, different quarantine objects, pay attention to the representativeness of different parts of the goods, and use diagonal, chessboard or random sampling methods to extract representative samples. The number of samples and the number of samples per sample are shown in Tables 2 and 3. Table 2 Number of samples taken on site
Total amount of goods
Less than 100 pieces
101~500 pieces
501~3.000 pieces
More than 3000 pieces
Plant species
Number of samples per piece
Tuber, root, onion, garlic, red date, etc. Peanut, corn, soybean, broad bean, kidney bean, etc. Seeds
Seedlings
Fruits
Rice, wheat, sorghum, mung bean, cotton seed, etc. Millet, millet, sesame, rapeseed, flax seed, etc. Vegetables, forage seeds, flower seeds, etc.
Tobacco seeds, etc.
Citrus, apple, flowers, potato seedlings, etc.
Citrus, apple, etc.
Note: For other categories, refer to the types in the table for sampling. 276
Number of samples
Number of samples per sample, g or plant
200~2500
1000～1500
10~100
2000~2500
6.1.3 Extraction of test samples
6.1.3.1 Seeds
GB 15569-1995
The representative samples taken on site shall be fully mixed and spread on a glass plate with a sample thickness not exceeding 1cm. Then, the number of samples required for indoor testing shall be extracted by the square quartering method. 6.1.3.2 Samples taken on site for seedlings, fruits, tubers (stems) shall be inspected one by one. Those with pests and symptoms of diseases and insect damage shall be inspected for indoor internal inspection and microscopic examination. 6.2 Instruments and Equipment
Instruments and equipment used for quarantine inspection of agricultural plants for transportation are shown in Appendix Q (reference). 6.3 General Indoor Inspection Methods
Plant quarantine personnel shall adopt one or more inspection methods according to the conditions of the items to be inspected. 6.3.1 Seed Sieving Inspection
Pour the sample to be inspected into the sieve with the corresponding aperture. The sieve hole specifications are shown in Table 4. Table 4 Sample Types and Sieve Hole Size Specifications Table Sample Types
Peanuts, soybeans, corn, comb seeds, etc. Rice, wheat, sorghum, hemp seeds, etc.
Millet, rapeseed, sesame, flax seeds, etc. Calculate the content according to the following formula when necessary.
Number of sieve layers
Specifications of apertures of each layer, mm
3.5, 2.5, 1.5
2.5, 1.5
2.0, 1.2
Content per kilogram:
=Number of found seeds (g)/sample weight (g)×10006.3.2 Seed specific gravity test
Use the specific gravity difference between diseased seeds, insect seeds and healthy seeds, use solutions of different specific gravity to distinguish between sinking and floating, and then pick up the floating seeds for further inspection.
6.3.3 Seed dyeing test
6.3.3.1 Potassium permanganate dyeing method
Mainly used to test cereal seeds.
Take 15g of clean sample and put it into a metal mesh or plastic mesh, soak it in 30℃ warm water for 1min, transfer it into 1g potassium permanganate solution and soak it for another 1min, take it out and immediately rinse it with clean water for 20~30min until it is clean, check it under a magnifying glass, and pick out the seeds with black spots of about 0.5mm on the surface for further inspection.
6.3.3.2 Potassium iodide staining method
Mainly used to inspect bean seeds.
Put 50g of sample into a metal mesh or plastic mesh, put it into 1% potassium iodide solution or 2% iodine tincture for 1~1.5min, transfer it into 0.5% sodium hydroxide or potassium hydroxide solution and soak it for 20~30s, take it out, rinse it with clean water for 0.5min, and check it with a magnifying glass or naked eye, and pick out the seeds with black spots of 1~2mm on the surface for further inspection. 6.3.3.3 Oil immersion inspection method
Mainly used to inspect bean seeds.
Use 1-1.5ml of olive oil, vaseline or machine oil for 1g of bean.5mL, mix well, soak for 0.5h, then the beans soaked in oil will turn amber, and the larvae's holes will show transparent spots.
6.3.4 Direct microscopic examination
Identify the species of the seized pests under a binocular dissecting microscope, pick the lesioned part of the sample, smear it on a glass slide, and check the pathogen species under a microscope.
6.3.5 Washing test
GB155691995
Pour 5~~~25g of the sample to be tested into a triangular flask with 10~50ml sterile water, shake for 5~10min, concentrate by centrifugation, take the concentrated precipitate and dilute it appropriately, check the pathogen species under a microscope, and calculate the load of the pathogen inoculum if necessary. 6.3.6 Anatomical examination
For agricultural plants and their products that are subject to inspection and are infected with difficult diseases or hidden pests, use a knife to dissect the damaged part or slice it, and then examine it under a dissecting microscope or microscope.
6.3.7 Isolation and culture test
Disinfect the sample to be tested, and transfer it to the corresponding culture medium for culture test. 6.3.8 Serological reaction test
Use special antiserum to test bacteria, viruses, and mycoplasmas respectively. 6.3.9 Bacteriophage test
Use the infection test of specialized phage to quickly test bacteria. 6.3.10 Sprouting test
Commonly used test methods include moisturizing culture, SARS sprouting, sprouting in soil, and observation of test tube seedling symptoms. 6.4 Test methods for several major quarantine objects The following quarantine objects shall be tested according to the following methods, and the inspection of other quarantine objects and supplementary quarantine objects shall be carried out in accordance with the provisions of the state and relevant provinces (autonomous regions, municipalities directly under the central government). 6.4.1 Inspection method for rice bacterial leaf streak [see Appendix D, (Supplement) 6.4.2 Inspection method for wheat dwarf bunt [see Appendix E, (Supplement) 6.4.3 Inspection method for cotton verticillium wilt [see Appendix F, (Supplement) 6.4.4 Inspection method for citrus canker [see Appendix G, (Supplement) 6.4.5 Inspection method for potato cancer [see Appendix H, (Supplement) 6.4.6 Inspection method for sweet potato blast [see Appendix J, (Supplement) 6.4.7 Identification of wheat Hessian mosquito Methods [See Appendix K, (Supplement) 6.4.83
See Appendix 1, Supplement) Identification methods for citrus fruit flies [See Appendix M, (Supplement) 6.4.9
See White moth identification methods [See Appendix N, (Supplement) 6.4.11 See Appendix P, (Supplement)] 7 Assessment and certification
7.1 Assessment
Evaluate the inspected agricultural plants and their products based on the results of on-site or indoor inspections. 7.2 Certification
Issue plant quarantine certificates and release if no quarantine objects are found, and no certification will be issued for re-inspection; if plants or products carrying or infected with quarantine objects are found, in addition to retaining samples and specimens, issue a notification form for the inspection results of agricultural plant transportation quarantine [See Appendix C, (Supplement)], notify the reporting unit or individual, and immediately take measures such as disinfection, change of use, controlled use or destruction, and supervise the implementation.
Name of plant and product
Destination
Shipping unit (person)
Plant quarantine station:
Hereby introduce
and other plants and plant products
Special quarantine seal of issuing authority
Validity period:
(unit)
GB 15569-1995
Appendix A
Application form for quarantine inspection of agricultural plants for transportation
(supplement)
Packing materials and methods
Weight (number of plants)
Destination
Means of transport
Receiving unit (person)
Appendix B
Requirement for quarantine inspection of agricultural plants for transportation
(supplement)|| Comrade tt|| is going to
kilograms (plants), and the quarantine object is:
Quarantine officer
No.:
Applicant (stamp):
Contact person (signature):
Telephone:
Postal code:
Contact for transportation
) Inspection word
Seeds (seedlings)
Year, month, day
Note: This form has three copies. The first copy is submitted to the quarantine authority of the place of transfer, the second copy is sent to the quarantine authority of the province of transfer, and the third copy is retained by the issuing authority. 279
Recipient
Sample name
Inspection purpose
Inspection method and result
Handling opinion
Special quarantine seal of issuing authority
GB15569--1995
Appendix C
Notification of inspection result of agricultural plant transportation quarantine (supplement)
Agent
Date of receipt
Number of samples
Date of inspection
Note: Two copies. The first copy shall be submitted to the inspection reporting unit (person), and the second copy shall be retained by the issuing quarantine authority. Appendix D
Test Method for Rice Bacterial Leaf Stripe Disease
(Supplement)
D1 Test Sample
)
Quarantine Officer
After the seeds are shelled, take 10g of husk, add 20mL of phosphate buffer PBS (pH7.0), soak overnight, filter to obtain the filtrate sample, and use one of the following three methods to detect the presence of bacteria.
D2 Bacteriophage Test Method
Take three sets of sterilized culture dishes, add 1mL of leaf stripe disease suspension (10°Cfu/mL) to each, and add 0.2, 0.5 and 1.0mL of sample filtrate to each, mix well, add 9mL of nutrient agar medium that has been entropy treated and cooled to 45-50℃ to each dish, shake well, cool, and place in a 28℃ constant temperature box for 12-18h to observe whether round transparent phage plaques appear. Result judgment: Seeds without plaques are disease-free seeds, and the number of plaques is related to the severity of the disease. D3 Direct separation method
Take 10mL of filtrate, centrifuge (8000r/min) for 10min, precipitate, resuspend in 1mL PBS, take 200μL and apply it on the pre-prepared nutrient agar plate. Repeat 10 culture dishes. Culture in a constant temperature box at 26-28℃ for 3-7d, and observe whether there are typical colonies with round, raised and uniform honey yellow texture.
D4 Serological test method
GB15569-1995
There are many test techniques to choose from, the most commonly used are enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), reverse indirect hemagglutination technique and Staphylococcus aureus coagulation reaction. The double antibody sandwich method is commonly used in enzyme-linked immunosorbent assay, see the test method for citrus canker [Appendix G, (Supplement)] for details; the reverse indirect hemagglutination method can be found in Appendix J (Supplement). The coagglutination reaction of Staphylococcus aureus is relatively simple and easy. Take a drop of concentrated sample suspension and place it on a glass slide, add a drop of Staphylococcus aureus sensitized by antibodies, and stir with a sterile toothpick or a thin bamboo needle for 30~~50s. If flocculent agglutination occurs, it indicates a positive reaction, otherwise it is a negative reaction. Sterile water or PBS solution is used as a negative control. Appendix E
Test method for wheat dwarf bunt
(Supplement)
Extract two samples of wheat samples by quartering, each weighing 50g, and pour them into a triangular flask respectively. Add 100mL of distilled water and shake vigorously for 5~10min. Then pour all the washing liquid into a clean centrifuge tube, centrifuge at a speed of 1000r/min for 5min, and pour out the supernatant. Add 1 mL of Shear's solution to the sediment at the bottom, shake the centrifuge tube, and use a clean pipette to draw the suspension onto a glass slide. Five slides are examined under a microscope for one sample. When identifying winter spores of dwarf stinking spores, 25 to 30 spores must be measured for each sample. The size of the winter spore mesh, the height of the mesh ridges, and the thickness of the gelatinous sheath are measured. If more than 70% of the spores have a mesh size of 3.5 to 5.0 μm, a mesh ridge height of 1.5 to 2.5 μm, and a gelatinous sheath thickness of 2.0 to 3.0 μm, they are determined to be dwarf stinking spores. The difference between dwarf stinking spores and common bunt spores is shown in Table E1. Table E1 Comparison of morphological characteristics of three wheat bunt pathogens Item
Mesh size, um
Ridge height, um
Gel sheath thickness, um
F1 Disinfection
Grain-shaped
Appendix F
Grain-shaped or nearly round
Below 1.0
Test method for cotton wilt
(Supplement)
Round and firm
Put cotton seeds in 70% alcohol for 2-3 seconds, remove bubbles, and place them in 0.Disinfect in 1% mercuric chloride solution for 2 minutes, rinse with sterilized water 2-3 times; soak cotton seeds in 20% bleach solution for 3-5 minutes, rinse with running water, place the seeds to be tested in a triangular bottle (500mL triangular bottle can hold 50-100 seeds), and cover the bottle with iron mesh or gauze. Take a section of rubber tube, connect one end to the tap, connect the other end to a glass tube, insert it into the angle bottle, turn on the tap, and rinse for 24 hours. 281
F2 separation
GB15569—1995
Place the sterilized seeds on the agar medium for separating Verticillium wilt and the PDA plate for separating Fusarium wilt, place 5~10 seeds per plate, place them in a 22~25℃ incubator for 3~4 days, then put them in a refrigerator for freezing (0~5℃, 24h), and then return them to the original incubator for about 10 days. Use a low-power microscope to observe the morphology of the macrospores to check the cottonseed fusarium; for Verticillium wilt, observe the whorl of small meristems and microsclerotia, and use a high-power microscope for further identification, and record the infection rate of the two pathogens. The number of seeds to be separated should be more than 1,000. It is also important to purify the isolated pathogens and inoculate them back to the roots of cotton seedlings to observe whether they are sick before making a conclusion. Appendix G
Testing Methods for Citrus Canker
(Supplement)
G1 Visual Inspection
G1.1 Branch Symptoms: The lesions are nearly circular, gray-brown, with a rough, raised surface and no yellow halo. Several lesions often connect into irregular patches. Under dry conditions, the canker lesions are spongy, corky, raised, and cracked on the surface; when wet, the cankers expand rapidly, with an intact surface and oily edges. Resistant varieties form a callus layer at the junction of diseased and healthy tissues. Canker disease can be confirmed by cutting off the external cork-like material with a knife and leaving a rough surface. Bright to dark brown lesions can be seen in dark green healthy tissues, and the size, shape, and depth of the discolored area vary. G1.2 Leaf Symptoms: The lesions are initially needle-sized, yellow, and oily. After expansion, both the front and back of the leaves are raised and cracked, spongy, and gray-white. Later, the diseased part becomes corky, with a rough surface and gray-brown crater-like cracks. The lesions are mostly nearly circular with yellow halos around them. The yellow halos on the lesions on old leaves are sometimes not obvious.
G1.3 Fruit symptoms: The lesions are similar to those on the leaves, but the crater-like cracking is more obvious, the degree of corkification is higher, and they are hard and rough. Generally, there is no yellow halo. The diseased part is limited to the peel and does not develop into the flesh. The lesions that occur in the early stage of fruit growth are mostly raised, while those that occur in the middle and late stages are relatively flat.
If the G1 inspection cannot be confirmed, the plant quarantine personnel shall adopt one or more of the following methods for inspection according to the situation. G2 Separation
G2.1 Take a small piece of diseased tissue, rinse it with sterile water, put it in 0.5-2.0mL sterile water, grind it with a sterilized glass rod, soak it at room temperature for 15-20 minutes, and separate the extract by streaking on the nutrient agar medium. If no symptoms are seen, wash the leaves with sterile water (more than 10 leaves), centrifuge and concentrate, make appropriate dilutions, culture on 28℃ artificial culture medium (more than 20 culture III lines), and pick single colonies. G2.2 The suitable isolation medium is NGA (3.0g beef extract, 5.0g protein Chen, 2.5g glucose, 16.0g agar). G2.3 On the culture medium of citrus canker bacteria, the colonies are round, yellow, shiny, whole, slightly raised, and sticky. The bacteria are short rods, often connected in chains, with a size of 0.50.7um×1.5～2.0um, round at both ends, with a single flagellum at the pole, can move, with capsules, no spores, and Gram-negative staining. G3 In vitro leaf enrichment
Collect the young top leaves of the susceptible citrus seedlings grown in the greenhouse, rinse with tap water for 10 minutes, disinfect the surface with 1% sodium hypochlorite solution for 1-4 minutes, rinse thoroughly with sterile distilled water under sterile conditions, then puncture the back of the leaf to create a wound, put the back side up in the culture III containing 1% water agar, add 10-20μL of lesion water extract to each wound (5-10 pinholes), culture at 25-30℃ with light for 5-7 days, observe the reaction of the puncture wound, and usually form typical tissue cracking symptoms within a week. If it is necessary to isolate the pathogen, the method is as follows.
G4 Serological test
G4.1 Preparation of antiserum: Standard antiserum is provided by a specialized agency. 282
GB15569--1995
G4.2 Enzyme-linked ELISA: Diagnostic kits are provided by specialized institutions and tested according to unified methods. G4.3 Immunofluorescence test (IF): Diagnostic kits are provided by specialized institutions and tested according to unified methods. G5 Pathogenicity test
Use the agglutinable bacteria obtained by separation and culture or the positive strains identified by serology to inoculate the seedlings of the diseased citrus trees and finally identify the citrus canker. The following three methods can be selected as one.
G5.1 Use a 25G hypodermic needle to inject and inoculate on the back of the detached leaves that have just turned green. The inoculation source concentration is 10%~10°Cfu/mL, and the culture conditions are the same as the detached leaf enrichment method.
G5.2 Use 170kPa pressure spray inoculation or needle inoculation to inoculate the leaves of the young shoots of the susceptible host seedlings, which are just beginning to turn green. The leaves that have just turned green are sharp. The inoculation source concentration is 107~10°Cfu/mL. Cultivate in a greenhouse and cover with a plastic cover to keep moisture for 24-72h. G5.3 Use the method of in vitro leaf enrichment to needle inoculate. Use diamond sand to scratch the in vitro leaves on the back side of the leaves, and inoculate with cotton balls saturated with the inoculation source. Appendix H
Testing methods for potato cancer
(Supplement)
H1 Tuber inspection
1.1 Cut the tuber tissue around the bud eye and make a cross-section with the skin, and examine the tuber cortex tissue for pathogens under a microscope. The pathogen is round and rusty in color, with a size of 50.4~81.9um×37.8~69.3um, thick wall, average 4.55um, layered, and irregular dormant spores on the outer layer.
H1.2 Slice the potato tuber or tear off a small piece of potato chip and place it on a slide, add a drop of 10% saffron dye solution, and observe under a microscope. If the cell wall is red, it is a healthy potato, and if it is dirty red, it is a diseased potato. H2 Diseased soil inspection (clean water floating method)
H2.1 Brush off the soil attached to the sample potato tuber carefully by batch, concentrate, and grind it finely. H2.2 Weigh the soil sample to be tested. Weigh 2~3 samples (depending on the number of soil samples) for each batch, and put each 3 into a polyethylene centrifuge tube (25mm×105mm) (glass centrifuge tubes cannot be used). Inject concentrated hydrofluoric acid (containing HF4851%) to half of the tube on a ventilation table, place it for 48 hours, and carefully mix the liquid in the centrifuge tube to promote the dissolution of silicon in the soil. H2.3 Carefully add distilled water to each centrifuge tube until the liquid level reaches about 13mm from the centrifuge tube mouth, and carefully stir the liquid. H2.4 Centrifuge at 2500~3000r/min for 10~15min, discard the supernatant, add distilled water to the original liquid level, stir well, centrifuge at the same speed for 10min, and then centrifuge 3~4 times. H2.5 Dilute the precipitate in the centrifuge tube and smear it, and examine it under a microscope for the presence of dormant cysts of this disease, and count the number of potato bacteria according to the dilution multiple and batch count.
Appendix
Testing methods for Gan Yi disease
(Supplement)
J1 Potato tuber symptoms
Potato tubers with mild disease have no obvious symptoms on the surface, only yellow-brown stains on the pedicle and tail root. Potato tubers with moderate disease have a small amount of yellow-brown lesions on the surface. After the pathogen invades, the vascular tissue of the tuber turns yellow-brown. When the tuber is cut crosswise, yellow-brown spots or plaques can be seen; when the tuber is cut longitudinally, yellow-brown stripes can be seen. Diseased potatoes have a bitter and smelly smell and cannot be cooked. Potato tubers with severe disease have brown water-soaked patches on the surface or are completely rotten. J2Burdon staining method
Wash the sample tuber, slice the vascular bundle of the suspected diseased tissue, directly smear the juice overflowing from the vascular bundle, heat and fix after drying, stain with Sudan black (B) for 5~~15min (the dye solution is 0.3g dye dissolved in 100mL70% ethanol), remove the excess dye, dry with absorbent paper, and after the smear is dried, re-stain with safranin (0.5% aqueous solution) for 5~10min, wash with water, and examine under a microscope after drying. J3 Indirect hemagglutination inhibition method
J3.1 Take a few grams of vascular bundle slices of the suspected diseased tissue and put them in a test tube of physiological saline (NaCl content 0.85%), let it stand for 30min to make an overflow solution.
J3.2 Take the antiserum (provided uniformly) and dilute it in multiples with 1:5 as the base, dilute to 1:5120 (the eleventh test tube), and arrange them on the 8×12 V-shaped agglutination plate in sequence. The twelfth column is arranged with physiological saline as the control. Use a standard dropper to drop one column for each concentration, one drop per hole (each drop is 0.025 mL).
J3.3 Arrange one sample in each row, set one physiological saline control, six materials to be tested, and one standard bacteria control, and drop one drop per hole, then shake it with a micro mixer for 2 minutes and place it in a 37℃ incubator for 40 minutes. J3.4 Add one drop of antigen-sensitized blood cells to each hole, shake for 2 minutes, cover the glass plate, and place in a 37℃ incubator for 90 minutes. J3.5 Result determination. The grading of the degree of hemagglutination is recorded according to conventional standards. In the hemagglutination inhibition test, more than ten ten ten are recorded as positive. The number of wells with inhibition is more than two wells more than the normal saline control, which is a positive reaction and is determined to be diseased; one well more than the control is a suspected positive reaction and suspected to be diseased; the same as the control is disease-free. Appendix K
Identification method of wheat Hessian mosquito
(Supplement)
Strip and inspect the inner side of the leaf sheath at the base of wheat straw, and sieve the seeds and the debris in the stacking site and transportation tools for inspection. The color, size and shape of the enclosure are similar to flax seeds, with an average length of 4.4mm, and some can reach 5.9mm. The front end is small and blunt, and the back end is large with a concave edge, in an asymmetrical rhombus shape. The enclosure contains white third-instar larvae or pupae. The third instar larvae are milky white and spindle-shaped, with a "Y\-shaped sword bone (thorax) on the ventral side of the prothorax, which is the main identification feature. The early stage is milky white, the middle stage is orange-red, and the late stage is dark brown. There is a pair of very short hairs on the front of the head; there is a long respiratory tube on the back of the thorax. Appendix L
Identification methods for apple aphids and apple capsule moths
(Supplement)
L1 Apple aphids
Each insect stage is spread with seedlings, scions, fruits, and transportation vehicles. Check the above parts and pay attention to tumors, nodes, scars, depressions, holes, etc. L1.1 Wingless adult aphid: The body is about 2mm long, oval, reddish brown, with hair warts on the sides of the body, and the back of the abdomen is covered with white waxy wool. The antennae have 6 segments, and the length is 1/4 of the body length. The third segment is the longest, which is roughly equal to the sum of the last three segments. The fifth and sixth segments each have a circular sensory circle, and the abdominal tube is degenerate. , leaving only slightly raised circular crack-like marks.
L1.2 Winged adult aphids: body length 1.7~～2.0mm, wings length 5.5mm, dark brown, antennae with multiple circular sensory circles on the 3rd to 6th segments. The forewings have 7 wing veins, the midrib has two branches, and the hindwings have 3 wing veins. There are fewer wax wool than wingless aphids. L1.3 Eggs: about 0.5mm long, 0.2mm wide, oval, slightly larger at one end, with prominent spermatophores, smooth surface, covered with white powder, initially laid with a yellow halo, then gradually turning brown.
GB15569-1995
L1.4 Nymphs: a total of 4 instars. In the first instar, it is flat oval, yellowish brown, and 0.65mm long; after the second instar, it gradually becomes conical and reddish brown; the antennae have 5 segments, the body length is 1.45mm in the fourth instar, and there is wax wool on the body. L2 Apple Wife Moth||t t||I.2.1 Larvae: Newly hatched larvae are white, with a light red or jade red back. Mature larvae are 14-18 mm long, with dark back and light belly. The head is yellowish brown, and the pronotum is light yellow with regular brown spots. The three bristles of the pronotum valve group are located on the same bristle plate, the three bristles of the first abdominal segment foot group are located on the same bristle plate, and the eighth abdominal segment foot group has two bristles. The anal plate is lighter in color than the pronotum, with small brown spots on it, no anal comb, and the hook of the abdominal foot toe is missing (missing outside). L2.2 Pupae: 7-10 mm long, yellowish brown, with a row of small spines on the back of the second to seventh abdominal segments, and only one row on the eighth to tenth segments. There are two hooked spines on each side of the anus, plus 6 at the end, for a total of 10. Appendix M
Identification Methods for Citrus Fruit Fly
(Supplement)||t t||This method is applicable to the inspection of citrus fruits.
M1 Check the outside of the fruit with the naked eye or a low-power magnifying glass for oviposition holes and fruit-dropping holes. M1.1 Oviposition hole: The oviposition hole is a small black dot in appearance, and the surrounding pericarp cells are tight and smooth, with a milky protrusion, sunken in the center, and surrounded by small gray-white cracks of cork. The fruit turns yellow before it is ripe. Use a blade to cut along the oviposition hole, and you can see that the white cortex is oil-soaked, with obvious halos, neat rings, and the petals are eaten dry (note that the difference from stink bug damage and mechanical punctures is that the latter's puncture points are not protruding or concave, and there are no halos on the white cortex when cut horizontally, and the petals are intact).
M1.2 Fruit-dropping hole: The hole is large inside and small outside, deep to the petals, and the outer edge is neat and smooth (note the difference from the puncture holes of adult noctuids: the puncture holes of noctuids are the same size inside and outside, and most of the edges are neat, and sometimes juice overflows. The difference from the holes of leaf roller larvae: the holes bored by leaf roller larvae are large on the outside and small on the inside, with gnawed edges and sometimes white filaments).
M2 larvae in the fruit after autopsy
Body length 15-17mm conical, small in front and large in the back, milky white and shiny. The front valve is fan-shaped, with a concave middle edge and about 30 finger protrusions (note that the difference from the larvae of the large orange fruit fly is that the front valve of the large orange fruit fly larvae is "T" shaped, slightly curved on both sides, with 33 to 35 finger protrusions, and the difference from the small fruit fly is that the front valve of the small fruit fly larvae is ring-shaped, with 10 to 13 finger protrusions), the rear valve is kidney-shaped, with 3 oblong cracks on the valve plate, 4 tufts of fine hairs on the outside (exposed tracheal tufts), and a button-shaped protrusion in the middle of the inside (note that the difference from the large orange fruit fly is that there are 5 tufts of fine hairs; the difference from the small orange fruit fly is that the rear valve is crescent-shaped, with 4 tufts of fine hairs on the outside, and the button-shaped protrusion on the inside is larger and more obvious). M3 adult identification
Body length 12~13mm (excluding ovipositor), wingspan 20~24mm. Yellowish brown. The back of the chest has bright yellow and dark brown stripes, often forming a "human" pattern in the middle. The chest has a pair of shoulder plate bristles, a pair of dorsal bristles in front and behind, two pairs of bristles on the hind wings, and a pair of small shield bristles. The abdomen is oval, with a black longitudinal stripe in the center of the back and a black horizontal stripe on the front edge of the third abdominal segment intersecting into a "cross" pattern. The female insect has 5 segments on the back, 3 segments for egg laying, the base segment is enlarged and equal to the abdomen, and the last 2 segments are narrow, but longer than the fifth abdominal segment (the main difference from the citrus fruit fly: the latter's ovipositor base segment is half the length of the abdomen, and the last 2 segments are shorter than the fifth abdominal segment). The male insect has 6 segments on the back, the last segment is short, and has a pair of long and S-shaped hooks. Appendix N
Identification method of American white moth
(Supplement)
Each insect stage has the possibility of spreading during transportation, among which the pupa is the most dangerous. N1 adults lie quietly in the corner of the carriage and on the bundle of seedlings. The adult head, thorax and abdomen are pure white, the compound eyes are dark brown to black, and the forewings R2, R3, R, and 2852 Pupa: 7~10mm long, yellowish brown, with a row of small spines on the back of the 2nd to 7th abdominal segments, and only one row on the 8th to 10th segments. There are 2 hooked spines on each side of the anus, plus 6 at the end, a total of 10. Appendix M
Identification method of citrus fruit fly
(Supplement)
This method is applicable to the inspection of citrus fruit.
M1 Check the outside of the fruit with the naked eye or a low-power magnifying glass for egg-laying holes and fruit-dropping holes. M1.1 Egg-laying hole: The egg-laying hole looks like a small black dot, and the surrounding pericarp cells are dense and smooth, with milky protrusions, sunken in the center, and corky gray-white small cracks around. The fruit turns yellow before it is ripe. Cut it horizontally along the egg-laying hole with a blade, and you can see that the white cortex is oily, with obvious halos, neat rings, and the petals are eaten dry (note that the difference from the damage caused by stink bugs and mechanical punctures is that the puncture points of the latter are not protruding or concave, and there are no halos on the white cortex when cut horizontally, and the petals are intact).
M1.2 Fruit removal hole: The hole is large inside and small outside, deep into the petals, and the outer edge is neat and smooth (note the difference from the puncture holes of the noctuid adults: the puncture holes of the noctuids are the same size inside and outside, and the edges are mostly neat, and sometimes juice overflows. The difference from the holes of the leaf roller larvae: the holes of the leaf roller larvae are large outside and small inside, with gnawed edges, and sometimes white filaments).
M2 Dissection of the larvae in the fruit
The body length is 15-17mm, conical, small in front and large in the back, milky white, and shiny. The front valve is fan-shaped, with a concave middle edge and about 30 finger protrusions (note that the difference from the larvae of the large orange fruit fly is that the front valve of the large orange fruit fly larvae is "T" shaped, slightly curved on both sides, with 33 to 35 finger protrusions, and the difference from the small fruit fly is that the front valve of the small fruit fly larvae is ring-shaped, with 10 to 13 finger protrusions), the rear valve is kidney-shaped, with 3 oblong cracks on the valve plate, 4 tufts of fine hairs on the outside (exposed tracheal tufts), and a button-shaped protrusion in the middle of the inside (note that the difference from the large orange fruit fly is that there are 5 tufts of fine hairs; the difference from the small orange fruit fly is that the rear valve is crescent-shaped, with 4 tufts of fine hairs on the outside, and the button-shaped protrusion on the inside is larger and more obvious). M3 adult identification
Body length 12~13mm (excluding ovipositor), wingspan 20~24mm. Yellowish brown. The back of the chest has bright yellow and dark brown stripes, often forming a "human" pattern in the middle. The chest has a pair of shoulder plate bristles, a pair of dorsal bristles in front and behind, two pairs of bristles on the hind wings, and a pair of small shield bristles. The abdomen is oval, with a black longitudinal stripe in the center of the back and a black horizontal stripe on the front edge of the third abdominal segment intersecting into a "cross" pattern. The female insect has 5 segments on the back, 3 segments for egg laying, the base segment is enlarged and equal to the abdomen, and the last 2 segments are narrow, but longer than the fifth abdominal segment (the main difference from the citrus fruit fly: the latter's ovipositor base segment is half the length of the abdomen, and the last 2 segments are shorter than the fifth abdominal segment). The male insect has 6 segments on the back, the last segment is short, and has a pair of long and S-shaped hooks. Appendix N
Identification method of American white moth
(Supplement)
Each insect stage has the possibility of spreading during transportation, among which the pupa is the most dangerous. N1 adults lie quietly in the corner of the carriage and on the bundle of seedlings. The adult head, thorax and abdomen are pure white, the compound eyes are dark brown to black, and the forewings R2, R3, R, and 2852 Pupa: 7~10mm long, yellowish brown, with a row of small spines on the back of the 2nd to 7th abdominal segments, and only one row on the 8th to 10th segments. There are 2 hooked spines on each side of the anus, plus 6 at the end, a total of 10. Appendix M
Identification method of citrus fruit fly
(Supplement)
This method is applicable to the inspection of citrus fruit.
M1 Check the outside of the fruit with the naked eye or a low-power magnifying glass for egg-laying holes and fruit-dropping holes. M1.1 Egg-laying hole: The egg-laying hole looks like a small black dot, and the surrounding pericarp cells are dense and smooth, with milky protrusions, sunken in the center, and corky gray-white small cracks around. The fruit turns yellow before it is ripe. Cut it horizontally along the egg-laying hole with a blade, and you can see that the white cortex is oily, with obvious halos, neat rings, and the petals are eaten dry (note that the difference from the damage caused by stink bugs and mechanical punctures is that the puncture points of the latter are not protruding or concave, and there are no halos on the white cortex when cut horizontally, and the petals are intact).
M1.2 Fruit removal hole: The hole is large inside and small outside, deep into the petals, and the outer edge is neat and smooth (note the difference from the puncture holes of the noctuid adults: the puncture holes of the noctuids are the same size inside and outside, and the edges are mostly neat, and sometimes juice overflows. The difference from the holes of the leaf roller larvae: the holes of the leaf roller larvae are large outside and small inside, with gnawed edges, and sometimes white filaments).
M2 Dissection of the larvae in the fruit
The body length is 15-17mm, conical, small in front and large in the back, milky white, and shiny. The front valve is fan-shaped, with a concave middle edge and about 30 finger protrusions (note that the difference from the larvae of the large orange fruit fly is that the front valve of the large orange fruit fly larvae is "T" shaped, slightly curved on both sides, with 33 to 35 finger protrusions, and the difference from the small fruit fly is that the front valve of the small fruit fly larvae is ring-shaped, with 10 to 13 finger protrusions), the rear valve is kidney-shaped, with 3 oblong cracks on the valve plate, 4 tufts of fine hairs on the outside (exposed tracheal tufts), and a button-shaped protrusion in the middle of the inside (note that the difference from the large orange fruit fly is that there are 5 tufts of fine hairs; the difference from the small orange fruit fly is that the rear valve is crescent-shaped, with 4 tufts of fine hairs on the outside, and the button-shaped protrusion on the inside is larger and more obvious). M3 adult identification
Body length 12~13mm (excluding ovipositor), wingspan 20~24mm. Yellowish brown. The back of the chest has bright yellow and dark brown stripes, often forming a "human" pattern in the middle. The chest has a pair of shoulder plate bristles, a pair of dorsal bristles in front and behind, two pairs of bristles on the hind wings, and a pair of small shield bristles. The abdomen is oval, with a black longitudinal stripe in the center of the back and a black horizontal stripe on the front edge of the third abdominal segment intersecting into a "cross" pattern. The female insect has 5 segments on the back, 3 segments for egg laying, the base segment is enlarged and equal to the abdomen, and the last 2 segments are narrow, but longer than the fifth abdominal segment (the main difference from the citrus fruit fly: the latter's ovipositor base segment is half the length of the abdomen, and the last 2 segments are shorter than the fifth abdominal segment). The male insect has 6 segments on the back, the last segment is short, and has a pair of long and S-shaped hooks. Appendix N
Identification method of American white moth
(Supplement)
Each insect stage has the possibility of spreading during transportation, among which the pupa is the most dangerous. N1 adults lie quietly in the corner of the carriage and on the bundle of seedlings. The adult head, thorax and abdomen are pure white, the compound eyes are dark brown to black, and the forewings R2, R3, R, and 285
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