Read the untranslated law here: https://www.vestnesis.lv/ta/id/113807
Cabinet of Ministers Regulations No. 568 Riga, 26 July 2005 (pr. No 43 41) potato brown rot of combat and control procedure Issued under the plant protection act, article 13 i. General questions 1. determines the order in which combats the potato ring rot in dark and limited its spread.
2. potato dark ring suggests the plant quarantine organism Ralstonia solanacearum (Smith) Yabuuc et al. (hereinafter body).
3. To establish the dark ring of potatoes, State plant protection service (hereinafter service): 3.1 regularly checks the potato Solanum tuberosum l. plants and tubers (potatoes) and eat tomato Lycopersicon esculentum Mill. (SYN. Lycopersicon lycopersicum (l.) H. Karsten) plants, other than fruit and seeds (hereinafter referred to as the host);
3.2. take seed and other potatoes for use of storage of samples and send them for laboratory testing;
3.3 check visually by cutting potatoes for Potato tubers. If it is found in the potato brown rot of potatoes of Visual signs, take samples and send them for laboratory testing;
UR3.4.izv ērtēj the risk of the organism spreading, check: 3.4.1. knockturn Solanace family au gus, including these native wild plants (hereinafter referred to as the other organism);
3.4.2. surface water used for irrigation and host plants of the organism for watering;
UR3.4.3.uz hospitality structures, dealing with the processing and packaging of host plants, as well as liquid waste used for the irrigation and spraying of host plants;
UR3.4.4.aug of substrate, soil and solid waste from the production host processing and packing plant;
3.5. check reservoir and taking water samples for laboratory testing, if the received information from another Member State of the European Union, in this country the infected host plants used for surface irrigation water that flows into Latvia;
3.6. diagnosed organisms in accordance with the provisions of the annex on Ralstonia solanacearum (Smith) Yabuuc et al. detection and identification of samples of potato tubers;
3.7. determines, controls and forcibly take control of the organism and control measures (hereinafter referred to as the phytosanitary measures).
4. a Person must not store or replicate the agent of the potato brown rot of pure cultures of the organism, except in the cases laid down in the legislation for trial or scientific purposes and for work on varietal selections of harmful organisms, plants, plant products and contact with them came the arrival of the items and the order.
II. determination of phytosanitary measures 5. If Visual inspection of potatoes service finds potato brown rot or signs, by laboratory testing in accordance with the provisions referred to in the annex of the Immunofluorescence Assay, a positive test result is a service shall decide on the following measures: a plant 5.1. prohibit the host Party or movement of goods from the farm, from which the sample was taken, unless the host is moved to the service of the organism have been eliminated and the risk;
5.2. at the risk of the organism spreading evaluation to prohibit a person from holding to move other farm grown host plants of the organism.
6. If the subsequent laboratory tests in accordance with the provisions set out in the annex to the methods of work at the laboratory, the body is not found, the Department under certain phytosanitary measures shall be repealed.
7. to determine the provenance of the organism, the service checks a lot of potatoes that have been associated with this provision referred to in paragraph 5 potatoes.
8. If two different provisions referred to in the annex to these laboratory tests are found in the body, the Department confirms the dark ring of potatoes.
9. If a body is found, the service shall decide on the application of phytosanitary measures. The decision includes this provision 10, 11, 12, 13, 14 and 15 of the type referred to in paragraph 1.
10. in order to determine the organism and mark the land plan in the infected area (fields, the place of production and the holdings that are in any way associated with the infected potatoes), infected States: 10.1 host plants and their cargo or the lot from which the sample was taken (hereinafter referred to as the infected host plants);
10.2. the inventory or parts, agricultural machinery and vehicle which had contact with the infected host plants;
10.3. other objects, including packaging, which had contact with the infected host plants;
10.4. the field, cover the area and the place of production from which the infected host plants for harvest, as well as the Notes field and place the parcel plan;
10.5. the field, cover areas and growing place where host plants growing in the period took samples of host plants, as well as the Notes field and the raising of the site plan;
10.6. other host plants of the organism from which the sample was taken;
10.7. the surface water from which the sample was taken.
11. in order to determine the organism and mark the land in the infected area, the plan for potentially infected States: 11.1. all host plants grown at the place of production, which sets the rules referred to in paragraph 10;
11.2. the place of production, which is a reference to the infected host plants, including those that are common in agricultural machinery, vehicles and rooms with growing site that is found in the infected host;
11.3. the host which has grown 11.2. these provisions in the place of production referred to in or after the capture of the organism was in the place of production, set to be infected;
11.4. warehouses in which are stored or processed in these rules the host from 11.2 in the above places of production;
UR11.5.iek ārt, agricultural machinery, vehicles, warehouse or parts and other items, including the packaging, which in the previous 12 months were able to come into contact with the infected SAIM niekaug;
11.6. host plants, which come in contact with this rule 11.5. the items referred to in point before cleaning and disinfection;
7.3. potatoes, which are linked to the infected lots of seed potatoes or tomatoes, origin, coming from the same place of production with infected tomatoes, if in accordance with the provisions laid down in point 6 of the inspection and test results, the body was not found, however, there is a risk of the organism spreading, plants reproduction;
11.8. the place where grown this rule 7.3. referred to Syme niekaug;
7.4. host plants growing on sites that use irrigation or spraying water, defined as contaminated;
11.10. host plants grown on fields flooded with surface water identified as contaminated.
12. the service determines the following: 12.1. phytosanitary measures prohibit planting infected and potentially infected host plants;
12.2. instructs you to do one of the following: 12.2.1. service of the burn the infected host plants;
12.2.2. infected host plants after heat treatment that destroys the body, used for animal feed;
12.2.3. the infected host plants of deep burial of waste dumps, from which the body is not able to get agricultural land for disposal, and no contact with water reservoirs, where water can be used for irrigation of agricultural land or watering;
12.2.4. industrial processing infected host plants, immediately bringing them directly to the processing plant, which has a suitable waste treatment facilities, warehouse and vehicle disinfection system. These requirements apply if the processing undertaking agrees to rework the infected potatoes;
12.2.5. host plants durum production waste (including rejected scale and bark) and any other solid waste products, which have been in contact with host plants: 126.96.36.199. buried in landfills, bringing the industry directly to the landfill and the way to avoid their loss and to the body would not be able to get agricultural land, as well as this site would not have contact with the water reservoirs, where water can be used for irrigation of agricultural land or watering;
12.2.6. host liquid production waste: 188.8.131.52. If they contain solid particles, before tipping the filter or put the solid particles to separate them, and other solid waste be buried or disposed of in accordance with this rule and section 184.108.40.206.220.127.116.11.;
18.104.22.168. before tipping it heat for at least adjust, if a minimum temperature of 70 ° C; 22.214.171.124. carrying out other phytosanitary measures against the organism into the agricultural land and reservoirs, where water can be used for irrigation of agricultural land or watering;
12.2.7. execute other phytosanitary measures where there is a risk of spreading the organism is eliminated;
12.2.8. potentially infected Potato tubers: 126.96.36.199. under the supervision of the use of the service for their own consumption or to send directly to sales outlets. Potato tubers are delivered packed at the point of sale, and may not be repackaged;
188.8.131.52. industrially processed, immediately bringing the potatoes directly to the processing plant, which has a suitable waste treatment facilities, warehouse and vehicle disinfection system. These requirements apply if the processing undertaking agrees to recycle potentially infected potatoes;
184.108.40.206. execute other phytosanitary measures where there is a risk of spreading the organism is eliminated;
12.2.9. other parts of host plants, including trunks and leaves: UR220.127.116.11.izn to īcin;
18.104.22.168. execute other phytosanitary measures where there is a risk of spreading the organism is eliminated.
13. the service instructs potato grower in the countryside and settled areas, places of production designated as contaminated, depending on the intensity of use of the field to select and execute one of the following: 13.1 if the phytosanitary measures apply five years after the capture of the organism: UR13.1.1.izn īcin to volunteer for potatoes, tomato plants and other host plants of the organism;
13.1.2. ban the planting of potatoes au gus and tubers, tomato seedlings and sow seeds of the genus Brassica plants and other host plants of the organism, which can spread the organism until the field is free of pārziemojuš potatoes for at least two of the next vegetation period;
13.1.3. the fifth year of production may be planted to certified seed potatoes for potatoes for consumption, processing or feed, where at least two previous periods of vegetation not found pārziemojuš potatoes, tomato plants and other host plants of the organism;
13.1.4. regular potato or tomato production year, subject to the authorisation of the plant, certified seed potatoes to plant seed or other types of potatoes (such as food, feed, processing);
13.2. If the phytosanitary measures shall apply for a period of six years: UR13.2.1.izn īcin to volunteer for potatoes, tomato plants and other host plants of the organism;
13.2.2. the first three periods of vegetation in the install and maintain black fallow land, pastures with frequent and low nopļaušan or intense over-grazing, sow the grass seed or the taking of crops;
13.2.3. fourth and fifth period of vegetation to plant plants that are not found in the maintenance and spreading the organism risk;
13.2.4. Sixth potato or tomato-growing season to plant certified seed potatoes or seed other types of potatoes (such as food processing, feed).
14. the infected area of the body, fields or on places of production other than those referred to in paragraph 13 of these regulations, subject to the change of the plant: 14.1 after the first vegetation period prohibit the capture of the organism to plant the tubers, plants, or other host plants of the organism and asking to destroy volunteer potato and tomato au gus, and other host plants of the organism, or permission to plant only certified seed potatoes potatoes intended for other purposes (e.g., for food , processing, animal feed production, if it is destroyed) all pārziemojuš potatoes, tomato plants and other host plants of the organism;
14.2. in the second period of vegetation after the infection discovery permission to plant certified seed potatoes seed extraction and other types of potatoes (such as food, feed, processing);
UR14.3.tre in the vegetation period after infection detection allows for production of seed and other types of potatoes (such as food, feed, processing) to plant certified seed potatoes or potatoes that are directly derived from certified seed potatoes which have been checked;
14.4. in each vegetation period laid down this rule 14.2. and 14.3. bottom point, asking to destroy volunteer potato, tomato plants and host plants of the organism.
15. the service determines the following: 15.1 the phytosanitary measures cover areas prohibit planting Potato tubers and plants, as well as other host plants of the organism, except for the production of potatoes used certified seed potatoes, sīkbumbuļ, or meristēmaug, which have been checked and are in service a complete replacement of the growing medium and cover the area and the cleaning and disinfection of equipment;
15.2. instructs the service of the person: UR15.2.1.att or disinfect īrī infected or suspect warehouses, agricultural machinery, vehicles and other objects, destroy or disinfect the infected and potentially infected host plants on in-contact packaging. After disinfection, these objects are no longer considered infected or potentially infected;
15.2.2. immediately after each of the organism and the next vegetation period allowed to the first year of production of potatoes in fields that were determined to be infected, take all potato production-related machinery and facilities, cover the area cleaning and, if possible, also for disinfection;
15.3. the infected area-growing sites – at least three years after the capture of the organism a person asked: 15.3.1. seed potatoes to harvest, store and move separately from other objectives (such as food, feed, processing) for potatoes;
15.3.2. make machinery, storage and production sites for cleaning and, if possible, also for disinfection;
UR15.3.3.vis au places of production designated as contaminated, use only certified seed potatoes.
16. in order to determine the possible spread of the organism and the original provenance of the organism, the service check: 16.1. potatoes, associated with contaminated batches of potatoes;
16.2. the growing site, which is close to the place of production which has been found in infected potatoes.
17. in order to determine the provenance of the organism, the service check: 17.1. other this provision mentioned in paragraph 10.1 below host plants cultivation related sites: au UR17.1.1.kur or tumor grown host plants, associated with the infected host plants;
UR17.1.2.kur au grown host plants or tumor that service monitored as suspected of being infected with the organism;
UR17.1.3.kur au or tumor grown potatoes originating in associated with potatoes at the place of production, set to be infected;
UR17.1.4.kur au host plants are grown and which is adjacent to the place of production, defined as infected, including breeding sites that share the use of agricultural machinery, vehicles and rooms with a farm, which is found in the infected host;
UR17.1.5.kas is used for the irrigation and spraying of surface waters, which is identified as contaminated or suspected of being infected;
UR17.1.6.kas is used for the irrigation and spraying of surface waters, which are also used in growing areas that have been identified as contaminated or suspected to be infected;
UR17.1.7.kas flooded or have been flooded with surface water is defined as contaminated or suspected of being infected;
17.2. surface water used for irrigation or spraying, or appludinājuš in the field or place of production designated as contaminated;
17.3. other wild solanaceous host plants.
18. If the service finds the original organism material, service to its original material, random, pre-basic seed or basic seed potato inspection, associated with contaminated batches of seed potatoes, and look forward to the laboratory for testing GUIs.
19. Service: 19.1. the decision shall specify the deadline of phytosanitary measures and added to the map of the infected area;
19.2. the monitor and control of phytosanitary measures;
19.3. the decision within the time limit laid down in the places designated as contaminated, after harvest, take the potato;
19.4. in irrigation and watering system check and, if necessary, shall determine the prohibition of the use of water, to prevent the risk of the organism spreading;
19.5. monitor the farms, warehouses and Potato tubers or tomatoes with the movements of the related undertakings, as well as production sites, which use the agricultural technique, together with the growing sites were detected in infected potatoes.
20. the infected area in service at least three the next vegetation period after detection of the infection made this rule 3.1. and 3.2. checks referred to in point.
21. the infected area where surface water is definitely about infected or potentially infected, service: 21.1. periodically carry out annual checks, taking water and solanaceous host plants and testing in accordance with the provisions of the methods referred to in the annex;
21.2. irrigation and watering systems and, where appropriate, define water-use restrictions to prevent spraying of host plants risk of the organism spreading. You can cancel the prohibition, on the basis of the annual results;
21.3. the watch collection with a host of materials related to the manufacturing or packaging companies that have discovered the organism infected liquid waste.
22. when potato breeder has not fulfilled the service specified phytosanitary measures and infected or potentially infected areas are planted infected or potentially infected or non-certified potatoes, service enforced chemically or mechanically destroyed potato plantations. With the destruction of potato related costs shall be borne by the producers of potatoes.
III. The European Commission and the Member States of the European Union information
23. If, following the Visual inspection of potatoes service finds potato brown rot or signs in accordance with the provisions referred to in the annex of the immunofluorescence test (testing laboratory) of a positive test result and suspect that the organism is infected host plants used for surface irrigation water, which flows from another Member State of the European Union or source to another Member of the European Union, the service in each of these cases shall inform the Member States of the European Union.
24. the service shall inform the European Commission and the Member States of the European Union for each case where it is established, and the body of the report indicates the following: 24.1. date in accordance with the provisions of paragraph 5 of a positive test result, sampling and date of capture of the organism;
24.2. the registration number of the holding for plant-health control exposed the plants and plant products circulating in the registry of parties, in which the place of production, set to be infected;
24.3. the infected host plants the shipment or lot attached to the phytosanitary certificate or plant passport number;
15.2. the infected seeds or other purposes (for example, food processing, animal feed) potato varieties and categories;
15.2. Description of the defined infected area and phytosanitary measures laid down therein;
24.6. these rules referred to in point 8 the test result (extract, prepared the slide immunofluorescence testing, infected Eggplant (Solanum melongen) material and pure cultures of the organism).
25. where the Department intends to apply other phytosanitary measures of infected and potentially infected potatoes for use, before service of the application shall inform the European Commission and the Member States of the European Union.
26. the service every year until 1 June shall notify the European Commission and the other Member States of the European Union in the previous year, the checks carried out and their results. On the same farm for planting potatoes selected checks, notifications sent every year until 1 September.
Informative reference to European Union directives, the regulations include provisions deriving from Council of 20 July 1998 Directive 98/57/EEC on Ralstonia solanacearum (Smith) Yabuuc et al..
Prime Minister a. Halloween farming Minister – the Minister of the environment r. vējonis Editorial Note: the entry into force of the provisions by 5 august 2005.
Annex a Cabinet of 26 July 2005, regulations no 568 Ralstonia solanacearum (Smith) Yabuuc et al. detection and identification of potato tuber samples i. test sample test 1 sample size is 200 tubers of the standard. You can also test the smaller samples.
2. Preparation of samples for testing: 2.1. Potato tubers, even wash and allow them to apžū;
2.2.ar disinfected scalpel to remove Peel potato heel end piestipr in youth and cut 3-5 mm large pieces of potato;
2.3. place the pieces of potato single-use container and pour with 40-50 ml volume of maceration buffer. It is recommended to cut tissue processed immediately. If immediate treatment is not possible, store the samples up to 72 hours to +4 ° C +10 ° C or up to 24 hours at room temperature;
2.4. sample incubate for four hours with 50 to 100 Shaker revolutions per minute;
2.5. decant the macerate and concentrate bacteria by centrifugation sample (10000g) 10 minutes + 4 ° C to + 10 ° C temperature;
2.6. the supernatant liquid, without sediment. The resulting precipitate 1.5 ml pellet buffer dissolve and divide the sample for further analysis: 22.214.171.1240 Im used the bacteria Ralstonia solanacearum (Smith) Yabuuc et al.;
126.96.36.1990 Im stored reference. Sample reference part adds 200 ml sterile glycerol, mix and store – 75 ° C and-85 ° C temperature;
2.7. during testing, the sample cut vascular and sediment suspension is stored at + 4 ° C to + 10 ° c.
II. test 3 immunofluorescence immunofluorescence (IF) test used for Multiwell microscope microscope slide. On slides with pencil inscription sample identification number.
4. Prepare the cake in pellet buffer decimal dilutions of 1/10, 1/100 and 1/1000 the tube with sediment mix. vortex. Pipette Lees and each dilution standard quantity-20 Italy – box line (table). The remainder of the line is used for duplicate or second sample.
Table Not diluted 1/10 1/100 1/1000 model 2 1 2 2 2 3 2 4 2 5 1 1 or 2 a duplicate sample sample 2 6 2 7 2 8 2 9 2 10 5. Prepare separate positive control slides (as in). Negative control used in pellet buffer. 5. The displacement and 10. priekšmetstikl eye undiluted. Each test kit uses a single slide.
6. the samples shall be dried until the slides are dry. The slides are incubated for 10 minutes at 96% ethanol and firing.
7. Prepare the antibody in IF buffer the working dilution, as indicated in the instructions of the manufacturer.
8. Cover the test boxes with the dilution of antibodies (antibodies used box quantity must be equal to the quantity of sediment used) and incubate for 30 minutes in humid Chamber at room temperature, if the recommendations of the manufacturer of the antibodies is not otherwise specified.
9. of the antimatter priekšmetstikl Harvest drops, and the slides carefully with IF buffer flush. Wash twice in five minutes in IF buffer. Carefully dry the excess moisture.
10. preparing IF buffer fluorescīnizotiocianāt (hereinafter referred to as FITC) conjugate dilution work, as indicated in the instructions of the manufacturer.
11. Cover the test boxes with FITC conjugate dilution (dilution of the conjugate box quantity must be equal to the quantity of dilution of the antibodies used) and incubate for 30 minutes in humid Chamber at room temperature if the manufacturer's instructions in the FITC conjugate unless otherwise indicated.
12. the Kicking of the glass slide drops of conjugate. Rinse and wash as above. Carefully dry the excess moisture.
13.Ar micropipette for each 5 drops of phosphate and glycerol Structural buffer or similar montējoš feature and cover with a coverslip.
14. The overview slide test boxes with filters for fluorescent microscope suitable for FITC, using oil immersion with a magnification of 1000 times. Viewing boxes across two diameters at right angles and around the perimeter.
15. First examine positive and negative control slides. Positive control cells must be bright fluorescent and completely painted. If they are not painted, repeat the test. Negative control boxes may not be the typical Fluorescing cells, otherwise the test repeated.
16. Looking at the slide test boxes typical Fluorescing cells with characteristic of Ralstonia solanacearum (Smith) Yabuuc et al. morphology. The fluorescence intensity must be the same as the dilution of positive control. Cells with incomplete staining or with weak fluorescence ignores. If such a cell is much based on the interpretation of the IF test result.
17. IF test result interpretation: 17.1. If bright Fluorescing cells with characteristic morphology are not found, the IF test is negative;
17.2. If bright Fluorescing cells with characteristic morphology are found, determine the mean number of cells in the ocular lens area and calculate the number of fluorescent cells (N) per ml of resuspended Pellet. On the border IF test is considered to be approximately 103 cell population of re-suspended pellet per: 17.2.1. samples with N greater than 103 cells per ml of resuspended Pellet, the IF test is considered positive;
17.2.2. samples with N less than 103 cells per ml of resuspended Pellet, the IF test is considered positive as possible;
17.3. If you see a large number (> 105 cells per ml) of incomplete or weakly Fluorescing cells, the IF test is considered positive as possible.
III. PCR test 18. Bacteria DNA extraction: 18.1. place Im 220 lizēšan buffer into an Eppendorf tube;
18.2. the sample extract 100 Im;
18.3. the sample is incubated at 95 ° C for 10 minutes;
18.4. further incubation of sample on ice for 5 minutes;
18.5. add 80 Im lizozīm concentrate;
18.6. incubate at 37 ° C for a sample of 30 minutes;
11.6. the Structural solution of A 220 EasyDN, inverts the sample;
12.8. incubate at 65 ° C for 30 minutes;
11.7. Add 100 EasyDN Im solution B, inverts the sample until the formation of viscous solution;
18.10. Add chloroform Italy 500 and shake well;
18.11. centrifuge the sample (15000 g) 20 minutes at + 4 ° C;
18.12. the upper phase into a new microvial;
18.13. Add 1 ml of chilled (-20 ° C) 96% ethanol;
18.14. centrifuge the sample (15000 g) 20 minutes at + 4 ° C;
18.15. decant the liquid phase;
18.16. Add 500 Im cold (-20 ° C) 80% ethanol;
18.17. centrifuge the sample (15000 g) 20 minutes at + 4 ° C;
18.18. decant the liquid phase;
18.19. dry the precipitate at room temperature;
18.20. precipitate dissolves 100 Im sterile distilled water;
18.21. incubate the samples at room temperature for 20 minutes;
18.22. moved sample-20 ° C for further storage.
19. The PCR test progress: 19.1. prepare the PCR reaction mixture;
19.2. PCR reaction tube inserts 20 Structural reaction mix;
19.3. Insert 5 each tube test sample of Italy. Each sample shall be analysed;
19.4. the samples are placed in termoprocesor.
20. The PCR product into agarose gel: 20.1. prepare mixture and pour the agarose gel;
20.2. the sample is added to the Structural loading sample buffer 1;
20.3. oblige the 12 Im gel PCR product;
UR20.4.mal upper gel inserts 3 Structural holes 100 base pairs (bp) of DNA markers;
20.5. the distribution of the electric field of the PCR products with strength and power 65m voltage 130 V 60-80 minutes;
20.6. move the gel ethidium bromide solution and the adjust colors;
20.7. placed on the gel and transiluminator seen in the PCR products by UV light.
21. the data obtained in the PCR test interpretation: 21.1. If the negative control is not detected by the appropriate hole in the bp length of PCR Amplicon, the PCR test is considered negative;
21.2. If the hole identifies the fragment length under positive control signal, the sample is considered positive.
IV. Biological identification and pathogenicity test test 22. Tomatoes grown in greenhouses + 22 ° C and + 28 ° C and watered every day. Grafting tomatoes take third stage of development of the page. Important to infect as newer plants are more susceptible to infection than small parents, indicator.
23. One sample having 20 plants (10 plants, five plants and five positive control negative control plants). On the potty with a tomato plant type sample identification number and indicate whether it is a plant of positive control or negative control plants.
24. start Grafting with negative control plants, the pot at the end of the sample and positive control plants.
25. the Pot on the stem between the cotyledons and the first leaf. The day before grafting desirable plants without water. Stem ieskrāp-0,5 1,0 cm long and three quarters of depth. With a syringe or a pipette pipette sample stems and aizlipin with paraffin to dries. The same Act also with control plants.
26. Positive and negative control plants places separately from the sample.
27. at the beginning of the week the regular observation of the symptom-the plant Wilts, wasting and hloroz.
28. the identification of test progress: 28.1. further testing having plants with symptoms or the stem above the infected sites, disinfected with 70% ethanol, cut into small pieces, cover with sterile water, leave for 15 minutes, then extract the IF and PCR tests carried out;
28.2. If after four weeks of symptoms is observed plant by IF and PCR tests. 1 cm of the stem take part (above the infected sites) of all plants, disinfected with 70% ethanol, cut into small pieces, cover with sterile water and leave to stand for 15 minutes, then extract the IF and PCR tests carried out;
17.6. If you need additional pathogen recontamination from tomato, testing plants with symptoms or take the stem above the infected sites, disinfected with 70% ethanol, cut into small pieces, cover with sterile water and leave for 15, strapped on a yeast peptone glucose agar, incubated at YPG + 27 ° C to + 28 ° C. Colonies are observed each day, its plant for three days. Incubation can be observed up to six days. The colony is a Pearly glow, flat, irregular. Grown colonies and with pure cultures of dressing (diluting the colony with pellet buffer, 1Ķl colony, taking the loop with 1 ml) IF. (IF instead of having the test sediment extracts from the colonies.)
29. Also performs the following tests: UR29.1.kr āsošan by gramme (Ralstonia solanacearum (Smith) Yabuuc et al.);
29.2. oxidase test (Ralstonia solanacearum (Smith) Yabuuc et al.).
30. The organic pathogenicity test and test result interpretation: 30.1. where bio-pathogenicity test specimen is negative (no symptoms), but positive control is positive, the negative control is negative and IFA and PCR tests are negative, the sample is considered negative;
30.2. where bio-pathogenicity test specimen is negative (no symptoms), but positive control is positive, the negative control is in the negative, and IF and PCR tests are positive, the sample is considered positive;
30.3. If the biological specimen pathogenicity test is positive (symptoms), positive control is positive, but negative control is negative and IFA and PCR tests are positive, the sample is considered positive;
18.9. If confirmatory tests (IF, PCR) results do not match, perform re withdrawal of pathogen of tomato;
5. If you are typical of Pseudomonas solanacearum colonies and IF the test is positive, the sample is considered positive;
If there is no typical 30.6. Pseudomonas solanacearum colonies and IF test is negative, the sample is considered negative.
31. After the screening test – IF the test and PCR test-sample identification card indicates if found Pseudomonas solanacearum. If Pseudomonas solanacearum are found, the article added, "Testing continues using organic pathogenicity test and identification tests" and the final opinion on the sample provided by the test results.
V. Use solutions maceration buffer: 188.8.131.52.05 M PBS (PBS) buffer, pH 7.0;
Na2HPO4 4.26 g 32.2. –;
KH2PO4 2.72 g 32.3. –;
32.4. distilled water – 1000 ml dissolve ingredients and 32.5. check pH;
32.6. buffer solution sterilize autoclave + 121 ° C for 15 minutes.
33. Pellet buffer: 33.1.0.01 M phosphate buffer (PBS), pH 7.2;
33.2. Na2HPO4S12H2-2.7 g;
33.3. Na2HPO4S2H2-0.4 g;
20.8. distilled water – 1000 ml dissolve ingredients and 20.8. check pH;
20.9. buffer solution sterilize autoclave + 121 ° C for 15 minutes.
34. IF-buffer: 34.1.0.01 M phosphate buffer (PBS), pH 7.2;
21.3. Na2HPO4S12H2-2.7 g;
3. Na2HPO4S2H2 – 0.4 g;
NaCl 8.0 g 21.4. –;
34.5. distilled water – 1000 ml dissolve ingredients and 21.5. examine the pH;
21.6. buffer solution sterilize autoclave + 121 ° C for 15 minutes.
35. glycerol phosphate buffer: 35.1.0, 1 M phosphate glycerol (PGB) buffer, pH 7.6;
35.2. Na2HPO4S12H2-3.2 g;
35.3. NaH2PO4S2H2-0.15 g;
35.4. glycerol 50 ml; 35.5. distilled water, dissolve 100 ml; 35.6. ingredients and check pH;
22.2. buffer solution sterilize autoclave + 121 ° C for 15 minutes.
36. Lizēšan buffer: 36.1.100 mM NaCl;
36.2.10 mM TRIS-HCl (TRIS (hydroxymethyl) aminoetān) (pH 8.0);
36.3.1 mM EDTA (dihydrated disodium salt) (pH 8.0).
37. Lizozīm cleaner (50 mg/ml): 37.1.50 mg lizozīm;
37.2.1 ml in 10 mM TRIS-HCl. 38. Electrophoresis buffer: 38.1.10 times Tae (TrisAcetātaEDT);
38.2. TRIS base – 48.4 g;
23.8.-11.42 ml glacial acetic acid; 23.9. Edta – 3.72 g;
38.5. H2o-up to 1000 ml. 39. Agarose solution: 24.3.-1.5 g agarose;
39.2. once a Tae-100 ml. 40. PCR reaction mix: 24.9. sterile distilled water – 11.5;
40.2.10 xPCR buffer-2.5;
40.3.25-3 mM MgCl2;
40.7. Taq polymerase-0.5;
25.4. the sample-5.0. VI. medium used 41. Yeast peptone glucose agar: 41.1. the pH of 7.0 6.5 YPG from to;
41.2. yeast extract – 5.0 g;
41.3. proteoz – 5.0 g of peptone;
25.7. glucose-10.0 g;
VIII. PCR Primer sequences and used the software table 1 no PO box
The oligonucleotide primer sequence Amplicon length (bp) 1.
Oli-1 5 '-ggg gg the agc ttg CTA CCT gcc-3 ' 288 2.
Y-2 5 '-ccc Act GCT gcc tcc CGT agg AGT-3′ 2. table no PO box
The number of cycle length temperature (° C) objective 1.
1 2 min 96 Genomic DNA denaturation 2.
50 20 s 94 Fragment DNA denaturation of Primer hybridization 20 s 30 s 68 72 Fragment synthesis 3.
1 10 min 72 Fragments after completing the 4.
While the sample is located in the thermostat 4 storage testing materials IX. preservation and storage 49. If there is a suspicion of the presence of the organism, because in accordance with the method referred to in the annex are of a positive immunofluorescence test results, and the need to continue laboratory tests to confirm or not confirm the presence of the organism in a sample, the above test to complete save and be kept until completion of laboratory tests: 30.5. If possible , lot or part thereof (from which the sample was removed) in its original packaging with label;
30.6. If possible, all tubers or plants, of which remove samples;
30.6. any pellet stays and additional prepared immunofluorescence slides;
49. all relevant documentation.
50. If in the laboratory analysis in accordance with the methods referred to in this annex, the presence of the organism in a sample is approved, you must save and appropriate conservation of: 50.1. this annex 1 contains the material;
50.2. the infected tomato or Eggplant material inoculated sample with the tuber or plant extract;
50.3. The tirage liqueur for this distinction in the organism.
Minister of agriculture, Minister for the environment r. vējonis
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