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Delivery: <= 3 days. True-PDF full-copy in English will be manually translated and delivered via email. SN/T 2622-2019: Detection and identification of Xanthomonas axonopodis pv.citri Status: Valid SN/T 2622: Historical versions
Basic dataStandard ID: SN/T 2622-2019 (SN/T2622-2019)Description (Translated English): Detection and identification of Xanthomonas axonopodis pv.citri Sector / Industry: Commodity Inspection Standard (Recommended) Classification of Chinese Standard: B16 Word Count Estimation: 12,127 Date of Issue: 2019-09-03 Date of Implementation: 2020-03-01 Older Standard (superseded by this standard): SN/T 2622-2010 Regulation (derived from): Natural Resources Department Announcement No. 7 of 2019 Issuing agency(ies): General Administration of Customs SN/T 2622-2019: Detection and identification of Xanthomonas axonopodis pv.citri---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.(Method for quarantine and identification of citrus canker) ICS 07.10.99B15/19 People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard SN/T 2622-2019 instead of Sn/T 2622-2010 Method for quarantine and identification of citrus canker Published.2019-2009-03 2020-03-01 implementation Published by the General Administration of Customs of the People's Republic of China ????? ????, ????, ?? ForewordThis standard is prepared in accordance with the rules given in GB/T 1.1-2009. This standard replaces Sn/T 2622-2010 "Citrus quarantine identification method", the main changes compared with Sn/T 2622-2010 as follows. --- Revised the original standard quarantine identification and determination procedures for citrus canker bacteria; --- Added pictures of citrus canker bacteria; --- Added more alternative PCR methods such as the real-time fluorescence PCR detection method for citrus canker bacteria; --- Added the response of citrus ulcer pathogen determination. This standard is proposed and managed by the General Administration of Customs of the People's Republic of China. This standard was drafted. Chengdu Customs of the People's Republic of China, Shanghai Customs of the People's Republic of China. The main drafters of this standard. Xiang Yong, Chen Lingling, Fu Jingyuan, Yi Jianping, Shao Baolin, He Wanxing, and Wang Chenghua. The previous editions replaced by this standard are. --- SN/T 2622-2010. SN/T 2262-2019 ????? ????, ????, ?? Method for quarantine and identification of citrus canker1 ScopeThis standard specifies the quarantine and identification methods for citrus canker bacteria in entry and exit plant quarantine. This standard applies to the quarantine and identification of citrus canker bacteria in Rutaceae plants and their fruits.2 Normative referencesThe following documents are essential for the application of this document. For dated references, only the dated version applies to this document. file. For undated references, the latest version (including all amendments) applies to this document. GB/T 6682 Laboratory water specifications and test methods Sn/t 1157 quarantine regulations for entry and exit plant nursery stock Sn/t 2122 quarantine sampling3 Basic information on germsProteobacteria, gamma-proteobacteria Gammaprotobacteria, Xanthomonadalales, Xanthomonaceae4 Method principleHarmful symptoms, colony morphological characteristics, pathogenic response, and PCR-specific response of the pathogen are the main basis for its quarantine identification. ５ Major reagents Unless otherwise specified, the reagents in this standard are analytical reagents or biochemical reagents. For the test water, refer to GB/T 6682. 70% ethanol, peptone, beef extract, yeast extract, sucrose, agar, ethidium bromide (EB), agarose, DNA Marker, TaZDNA polymerase, PCR system, MgCl2, dNTP, Tayman probe, etc.6 EquipmentStereo microscope, super earning bench, autoclave, biological incubator, electronic balance, ordinary refrigerator, ultra-low temperature refrigerator, centrifuge, vortex Mixer, pure water meter, turbidimeter, PCR meter, real-time fluorescence PCR meter, electrophoresis meter, gel imaging system, micro-adjustable sampler, scissors, culture Petri dishes, triangle bottles.7 Quarantine and identification7.1 Quarantine and identification process The identification process of citrus canker is shown in Figure 1. SN/T 2262-2019 ????? ????, ????, ?? Figure 1 Identification process of citrus canker 7.2 Symptom check Perform on-site quarantine and take samples according to the procedures specified in Sn/T 1157 and Sn/T 2122 Laboratory symptoms Check to see if the sample to be tested has suspected symptoms (see Appendix A). If there are symptoms, cut out the tissues under the symptoms and observe whether under the microscope There is a spraying phenomenon. If there is a spraying bacteria, isolate the bacteria directly from the spraying tissue. If the sample is asymptomatic or no symptom is found in the sample, perform a preliminary PCR test. See Appendix B for the PCR method. Samples with positive PCR results Isolation of germs. 7.3 PCR detection Take asymptomatic or symptomatic samples without spraying bacteria, soak in sterile normal saline at room temperature for 30min or 4 ℃ refrigerator overnight, shake, and take The soaking solution was centrifuged at 10,000 r/min for 10 min, and DNA was extracted by precipitation. Routine PCR or real-time fluorescence PCR detection (Appendix B) Duck ulcer bacteria DNA was used as a positive control, non-citrus ulcer bacteria DNA was used as a negative control, and double distilled water was used as a blank control. 7.4 Isolation of germs A 2mm × 2mm piece of tissue was taken from the symptom of the symptomatic sample, and the incision was observed under the microscope at 10 × ～ 20 × times after making the slide Cloudy bacteria spewed from the incision. Cut 5 to 10 small pieces of tissue from each sample to observe spraying bacteria, and isolate 3 to 5 small spraying bacteria. SN/T 2262-2019 ????? ????, ????, ?? Block tissue, such as less than 3 sprayed tissue, can increase the number of microscopic examination of tissue. If spraying bacteria, remove the sprayed tissue pieces and rinse them with sterile water. 70% alcohol surface disinfection treatment for 30s to 60s, rinsed with sterile water three times, transferred to 100μL of sterile water and soaked for 15min; the treatment solution was separated on the NA plate, and 3 to 5 plates were marked for each treatment; Observe whether there are typical colonies after incubating for 2 to 4 days. Suspect After the colonies were purified three times, the next identification test was performed. If there is no evidence of bacterial spraying in the sample, take the tissue scissors of the symptom to cut into 2mm square pieces, and add appropriate amount of sterilized water to soak at room temperature. Soak in 30min or 4 ℃ refrigerator overnight, take half of the treatment solution at 1000r/min and centrifuge for 10min, precipitate and extract DNA, conventional PCR or real-time Preliminary fluorescence PCR test. The positive sample treatment solution was diluted 10 ×, 100 ×, and 1000 ×, and 100 μL of NA plate was taken, and 5 to 10 plates were coated for each treatment. The cultivation and observation methods are the same as above. If typical colonies are not isolated from PCR-positive samples, increase sample repetition. Or apply the treatment solution directly to the plate without dilution and observe the culture. For the initial examination and isolation of asymptomatic samples, refer to this method. 7.5 Identification 7.5.1 Colony morphological characteristics The colonies on the NA medium are smooth, opaque, shiny, round, raised, and the edges are intact; it is initially white and then turns light yellow; NBZ The colonies on the medium were light yellow, round, raised, and slime-like; the colonies on the PSA medium were pale yellow, mucus-like, and shiny. Pick Suspicious Bacteria After falling on the NA medium plate and purifying three times, it was subjected to biolog measurement or PCR identification. See Appendix A for the physiological and biochemical characteristics of the pathogen. 7.5.2 PCR amplification Suspect isolates were cultured on a NA plate for 48 h at 28 ° C. The bacterial cells were collected to extract DNA for routine PCR or real-time fluorescence PCR detection. Testing, see Appendix B for testing procedures. 7.5.3 Pathogenicity determination The suspected isolate was cultured at 28 ° C for 48 hours on NA, and then prepared with 108 CFU/mL bacteria suspension with sterile water. Grapefruit, Mexican lemon, citrus and sweet orange) young leaves, with sterile water as a negative control. The plant was grown at 28 ° C, and the leaves were observed after 7 days. Incidence. The pathogens were re-isolated from the tissues where the symptoms appeared, and compared with the original isolates to complete the pathogenicity test.8 Evaluation of resultsThe suspected isolate was obtained, and the biolog test was positive or the PCR test result was positive. The pathogenicity test showed typical symptoms. The product was detected as citrus ulcer; in other cases, it was determined that citrus ulcer was not detected.9 Sample storage and processingThe samples are properly stored after being registered and signed by the owner. The samples of citrus canker can be stored at 4 ° C for review. Sample protection After expiration of storage period, autoclave inactivation treatment. 10 Preservation and Handling of Strains The strain isolated from the sample and identified as citrus canker bacterium should be stored properly. Configured as 108CFU/mL bacteria suspension, add 15 to 30% glycerol is frozen at -80 ° C; the strains can also be lyophilized and stored at -20 ° C or -80 ° C for a long time. Autoclave Inactivation processing. SN/T 2262-2019 ????? ????, ????, ??Appendix A(Informative appendix) Citrus Canker Information A. 1 English name Citrus CankerDisease; Citruscanker; Bacterialcankerofices; Citrusbacherialcanker. A. 2 Scientific name Chinese name. Citrus yellow single citrus subspecies al. ,.1995. A, B, C, D and E strains. In 1989, Gabriel et al. Studied the restriction enzyme fragment length polymorphisms of chromosomal DNA in various strains. A strain (Asian strain) originated in Asia and has the strongest invasiveness. Most citrus varieties can be affected by it, but grapefruit, loquat and sweet orange Vulnerable and cosmopolitan. B/C/D strains are also called lemon strains. Among them, B strain was reported in Argentina in 1928, It causes disease spots on lemons and Mexican lemons, hardly develops on grapefruit and sweet oranges, and is mainly prevalent in South America. In 1972, the C line was discovered In Brazil, it develops on lemons and limes in Mexico, but not on grapefruit and sweet oranges. In 1981, the D strain was in Colima, Mexico. The district found that the pathogenicity was very weak, and its symptoms were similar to those of type A ulcers, but no fruit harm was reported. Mainly harms Mexican lemons, also SN/T 2262-2019 ????? ????, ????, ?? Its symptoms are significantly different from type A, B, C, and D ulcers. The leaf spots are water-stained, greenish and necrotic, but the tissue is not corked and bulged, and it is not broken. However, it does not harm the fruit under the conditions, and the invasiveness to grape sleeves is stronger. A. 3 Geographical distribution Asia. Afghanistan, Bangladesh (partially distributed), Cambodia, China, Christmas Island, Cocos Islands, Georgia (possibly), India, India Tunisia, Iran (partially distributed), Iraq (probably), Israel, Japan, South Korea, North Korea, Laos, Malaysia, Maldives, Myanmar, Nepal, Oman, Pakistan, Philippines, Saudi Arabia (partially distributed), Singapore, Sri Lanka, Taiwan, Thailand, Turkey, Afghanistan United Arab Emirates, Vietnam, Yemen (locally distributed). Africa. Algeria, Benin, Burkina Faso, Comoros, Congo, Côte d'Ivoire, Egypt, Ethiopia, Gabon, Gambia, Canada , Guinea, Kenya, Libya, Madagascar, Mali (restricted distribution), Mauritius, Mayotte, Mozambique, Reunion Island, Rodrigues, Senegal, Seychelles, Somalia, South Africa (possibly, report eradicated), Sudan, Swaziland, Tanzania (partial Distribution), Tunisia, Zimbabwe. Americas. Mexico, US (locally distributed), Bahamas, Belize, British Virgin Islands, US Virgin Islands, Costa Rica Canada, Cuba, Dominica, Dominica (possibly), El Salvador, Guadeloupe (France), Haiti, Honduras, Jamaica, Martinique, Netherlands Andres Islands (possible), Nicaragua, Puerto Rico, Saint Lucia (possible), Trinidad and Tobago (possible), Agen Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, Paraguay, Peru, Suriname, Uruguay (locally distributed), Venezuela. Europe. Albania, Croatia, Cyprus, Malta, Netherlands. Oceania. American Samoa, Australia (possibly, report eradicated), Fiji, Guam (possibly), Marshall Islands, Micronesia Islands, New Zealand (possibly, reported eradication), Northern Mariana Islands (possibly), Palau, New Guinea, Solomon Islands. Citrus ulcers have different pathogenic, serotype and hereditary strains, and are usually divided into the following strains. A strain (CBCD-A, Asian ulcer or true ulcer), distributed in most citrus growing areas except the Mediterranean Basin and the United States; B strain (CCD- B, pseudoulcer disease type), distributed in Agenyan and Uruguay; C strain (CBDCD-C, Mexican lemon type), distributed in Brazil and Paraguay; D bacteria Line (CBDCD-D) is distributed in Mexico; Line E (CBDBD-E, citrus bacterial leaf spot) is distributed in Florida, USA. The Asian type A strain is the most virulent and most widely distributed, infecting many hosts of the Rutaceae family. Although B strain also infects citrus hosts, However, the main infection of lemons in Argentina, Uruguay, and Paraguay may be related to the pathogenic A strain. In Brazil, the C line A. 4 Host range A. 5 Spread and spread Citrus canker can invade plants mainly through their stomata and wounds. The transport of citrus materials such as diseased seedlings, scion and fruit is SN/T 2262-2019 ????? ????, ????, ?? The main way for long-distance transmission, the field mainly spreads through wind and rain, pests, and sometimes the soil with bacteria can also spread the disease. A. 6 typical symptoms A. 6.1 Blade At the beginning of the onset, yellow or dark yellow needle-point oily spots appear on the back of the leaf, which gradually expands. Gradually bulging, showing a nearly circular, beige lesion. Later, the epidermis of the diseased part will crack and become sponge-like, with more prominent bulge, cork, rough surface and grayish white. Or taupe. The center of the lesion is sunken, surrounded by yellow or yellow-green halo rings. Some varieties often have brown glazed edges near the halo rings. To In the later period, the center of the lesion became a crater-like crack. The size of the lesions varies by variety, with larger lesions on sweet orange, navel orange and pomelo, citrus, tangerine and lemon The lesions are smaller, as shown in Figure A. 1 and A. 2. A. 6.2 shoots The lesions are nearly round, gray-brown, with rough surface, protrusions, and no yellow halo. Multiple lesions are often connected into a piece and are irregular. Ulcers in dry conditions Lesions are spongy, cork-like, raised, and ruptured on the surface; when wet, the ulcers rapidly expand, the surface is intact, and the edges are oily. Lesions occur in the branches No yellow halo. Necrotic flat lesions are produced on the twigs, sometimes with greasy edges, without yellow halos. A. 6.3 Fruit The lesions are similar to the symptoms on the leaves, but the crater-like cracking is more pronounced, the degree of cork is higher, hard and rough, and there is generally no yellow halo. It is limited to the peel and does not develop into the pulp, see Figures A.3 and A.4. The lesions that occur in the early stages of fruit growth are more protrusive, while those that occur in the middle and late stages are relatively flat. level. Juvenile fruit has resinous secretions, which cause early fruit drop when the disease is severe. (犪) Citrus back (犫) Lemon front Figure A. 1 Symptoms on leaves Figure A. 2 Pathogenicity test (left. sterile water; right. citrus canker) SN/T 2262-2019 ????? ????, ????, ?? (犪) grapefruit (犫) lemon Figure A. 3 symptoms on fruit Figure A. 4 Symptoms on Fruit (Lime) A. 7 Morphological characteristics Gram-negative, short rod-shaped, obtuse at both ends, ranging from 1.5 μm to 2.0 μm × 0.5 μm to 0.75 μm, extremely single flagella, capsular, no spores. The colonies on the NA medium were round, whole, honey yellow, shiny, smooth, slightly raised, and sticky, as shown in Figure A. 5. A. 8 physiological and biochemical characteristics It can liquefy gelatin, hydrolyze casein, and grow in 3% NaCl solution. It does not make litmus cow milk, does not produce hydrogen sulfide, and decomposes lakes. Powder, hydrolysis of aescin, methyl red test was negative, hydrogen peroxide reaction was positive, oxidase reaction was negative, urea decomposition test was negative Can not degrade nitrate and indole products, can decompose arabinose, glucose, mannose, galactose, trehalose to produce acid. Optimal temperature of germs is SN/T 2262-2019 ????? ????, ????, ?? 28 ℃ ～ 30 ℃, the lowest is 5 ℃ ～ 10 ℃, the highest is 35 ℃ ～ 39 ℃, the lethal temperature is 55 ℃ ～ 60 ℃ 10min, the optimum pH is 6.6. A. 9 Difference between X 犮 犮 and its approximate species The difference between Xcc and its approximate species is shown in Table A. 1. Table A. 1 Difference between X 犮 犮 and its approximate species English name CBC (citrusbacherialcanker) CBC (citrusbacherialcanker) CBX (citrusbacherialport) Citrus canker Geographical distribution Asia, Africa and Central and South America (Mexico, Brazil, Argentina, Uruguay and Paraguay) Florida, USA Host grapefruit, sweet orange, sleeve, lemon, citron lemon, lime, mexican lime orange, tangerine, grapefruit symptom The initial stage is a herniated small herpes spot, and later Cork formation Spot; sometimes the glaze circle on the edge of the lesion is obvious, Peripheral water stain The initial stage is a small herpes spot. Embolization, forming rough raised lesions; disease Watermark Lesions are flat or sunken, with obvious edges Water stain Culture shape (28 ° C) Single bacteria on 40 ~ 44h on YDC Fall; no water-soluble pigments are produced. FS Single colonies were produced on 48-52h. 56 ～ 60h generate order on msX Colony Single bacteria on 56 ~ 60h on YDC Fall; can produce water-soluble pigments. FS A single colony was produced at 70-76 h. 80 ～ 84h generate order on msX Colony Single bacteria on YDC for 30 ～ 4h Fall; no water-soluble pigments are produced. FS Single colonies were produced in the last 40 to 44 h. Generate orders on the MSX 48-52h Colony Physiological and biochemical characteristics Available maltose and arabinose; Cannot utilize inulin; hydrolysable litmus Milk and casein Cannot use maltose; precipitated stone Rui milk; can not hydrolyze casein Available with maltose and inulin; Cellobiose and mannitol acid production Phage response (CP1 and CP2) sensitive not sensitive not sensitive Serological response (Citrus subspecies) positive, but different from Xcc negative SN/T 2262-2019 ????? ????, ????, ??Appendix B(Normative appendix) Citrus canker bacteria PCR method The primers are Xac01 (5'-cgcccatcccccccaccaccacc-3 ')/Xac02 (5'-accccctccatccccctcctc-3'); amplification For 581bp. The PCR reaction program was 94 ° C for 5 min; 94 ° C for 30 s, 60 ° C for 30 s, 72 ° C for 60 s, and 30 cycles; 72 ° C for 10 min. The primer is J-RTphth3 (5'-acccctccctctacacctcaa-3 ')/J-RTphth4 (5'-ccgccctcgagactgtc-3') and the probe is J- Tajphth2 (5'-FAM-atgccgccccccaccccc-TAMRA-3 '). Real-time fluorescence PCR reaction program. 95 ° C for 10 min; then 15 s at 95 ° C, 60s at 60 ° C, 40 cycles. If there is still a significant amplification curve, the test result is judged to be positive, otherwise it is negative. SN/T 2262-2019 ????? ????, ????, ?? ......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of SN/T 2622-2019_English be delivered?Answer: Upon your order, we will start to translate SN/T 2622-2019_English as soon as possible, and keep you informed of the progress. The lead time is typically 1 ~ 3 working days. The lengthier the document the longer the lead time.Question 2: Can I share the purchased PDF of SN/T 2622-2019_English with my colleagues?Answer: Yes. The purchased PDF of SN/T 2622-2019_English will be deemed to be sold to your employer/organization who actually pays for it, including your colleagues and your employer's intranet.Question 3: Does the price include tax/VAT?Answer: Yes. 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