Read the untranslated law here: https://www.vestnesis.lv/ta/id/159073
Cabinet of Ministers Regulations No. 365 in Riga on 29 may, 2007 (pr. No 31 62) of potato ring rot and control procedure Issued under the plant protection act article 5 paragraph 13 i. General questions 1. determines the order in which combats the potato brown rot disease and light limited its spread. 2. potato light ring suggests the plant quarantine organism Clavibacter michiganensis subsp. sepedonicus (hereinafter body). 3. to establish the potato ring rot, light State plant protection service (hereinafter service): 3.1 regularly checks the potato Solanum tuberosum l. tubers and plants;
3.2. in seed potatoes and potatoes for other uses of Solanum tuberosum l. tuber and plant samples in storage areas and sent for laboratory analysis;
3.3. test of potato Solanum tuberosum l. samples visually by cutting the tubers. If you are found of potato ring rot, potato of Visual signs of having samples sent to the laboratory and testing;
3.4. in accordance with the provisions listed in annex 1 of the laboratory methods are diagnosed;
3.5. the isolation and cultivation of the organisms used in these rules referred to in annex 2 of the medium;
2.2. laboratory analyses used in these rules referred to in annex 3 buffer;
3.7. the body's infection is established in accordance with the provisions of annex 4;
3.8. the laboratory analysis carried out in accordance with these rules 5, 6 and 7 of the annex;
3.9. the application and control of potato ring rot from spreading and control measures (hereinafter referred to as the health measures). 4. A Person must not be stored or reproduced, except pure organism legislation for trial or scientific purposes and for work on varietal selections of harmful organisms, plants, plant products and contact with them came the introduction into and movement of these cases. 5. in order to limit the spread of potato ring rot, the person who cultivates potatoes every year renew the seed potatoes certified seed potatoes with 25% of the apstādām area. 6. A Person who cultivates potatoes, store the documents proving the purchase certified seed potatoes. II. determination of phytosanitary measures 7. If the potato Solanum tuberosum l. examination Department finds Visual of potato ring rot symptoms or in accordance with the provisions referred to in annex 1 of the immunofluorescence (IF) test (hereinafter referred to as the IF test) for the first positive test results, the Department shall take a decision on such a phytosanitary measures: 7.1 prohibits moved from farm and planted it in the party or holding the load, from which the sample was taken until you complete all required by these rules, the said laboratory tests. If there is no risk of the organism spreading, the service of the person carried out only this rule 14.2.1 14.2.2 14.2.3., and the bottom measures referred to in paragraph 1;
7.2. prohibit a person to move from holding other solanaceous plants (Solanaceae) (-host);
7.3. take steps to trace the emergence of the organism may cause. 8. If, following this rule referred to in annex 1. the laboratory tests, the body is not found in the sample service repealed certain phytosanitary measures. 9. to determine the provenance of the organism, the service checks a lot of potatoes that were associated with that provision referred to in paragraph 7 of the potatoes. 10. Service confirms the bright ring of potatoes if the organism is found in at least two different these rules referred to in annex 1. laboratory tests. 11. If the found body, service shall decide on the application of phytosanitary measures in accordance with the provisions of 12, 13, 14 and 15. 12. in order to determine the prevalence of the infection and the site 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: 12.1. potato Solanum tuberosum l. tuber or plant or lot from which the sample was taken (the-infected potatoes);
12.2. the inventory or parts, agricultural machinery and means of transport that had contact with infected potatoes;
12.3. other items, including packages, which have been in contact with infected potatoes;
12.4. field and the place of production from which get infected potato harvest;
12.5. the rural and growing place where potato vegetation period takes samples of potatoes. 13. in order to determine the prevalence of the infection and mark the land plan in the infected area for potentially infected States: 13.1. any Potato tubers or au gus grown or located in the infected area;
13.2. the land, growing space and space related to Potato tubers or plants, designated as contaminated, including growing site that agricultural machinery, vehicles and premises used in common with places of production which has been found in infected potatoes;
13.3. Potato tubers and plants which have been grown 13.2. This provision places referred to in the determination of the infection or have been;
13.4. production site or premises in which an activity with potatoes, which moved from that rule 13.1 and 13.2 the places referred to in subparagraph or premises;
13.5 equipment, agricultural machinery, vehicles, warehouses and other items, including the packaging, which in the previous 12 months were able to come into contact with infected potatoes;
13.6. Potato tubers or plants, which have been in contact with warehouses or other objects before cleaning and disinfection;
8.5. Potato tubers or plants, which are associated with the infected lots of seed potatoes originating in or get in one place with infected potatoes because of the possible spread of the infection, plants reproduction;
13.8. this rule 8.5. referred to the place of production. 14. the service shall phytosanitary measures: 14.1 prohibits planting infected and potentially infected potatoes;
14.2. instruct you to do one of the following: 14.2.1. service of destroying infected potatoes;
14.2.2. the industrial processing infected potatoes, immediately bringing them directly to a processing plant with appropriate waste disposal facilities, warehouses and vehicles departing disinfection equipment. These requirements apply if the processing undertaking agrees to rework the infected potatoes;
14.2.3. after heat treatment that destroys the body, infected potatoes use feed;
14.2.4. infected potatoes delivered to a waste disposal site, where there is no risk that pathogens can get into the environment;
14.2.5. burn infected potatoes;
14.2.6. destroy infected potato waste and other solid waste associated with the potatoes, the infected waste disposal site at which there is no risk that the body would get to the environment, or burn, or take other measures, if the plant is mitigated risk organism spreading;
14.2.7. service of destroying infected potatoes with associated liquid waste;
14.2.8. the monitoring of the use of the potentially infected potatoes for other purposes (such as food or send directly to the sales outlets). Potato tubers are delivered packed at the point of sale, and may not be repackaged. Potatoes intended for planting may Pack just after the packing space cleaning and disinfection;
14.2.9. service of destroying potentially infected plants;
14.2.10. industrial processing potentially infected potatoes, bringing them immediately packed directly in the processing plant, which has a suitable waste disposal facilities and where possible disinfection of both vehicles. 15. the potato grower asked places of production designated as contaminated, choose and perform one of the following: 15.1 the phytosanitary measures in fields defined as contaminated, according to the intensity of use of the field shall apply one of the following: 15.1.1. phytosanitary measures if the phytosanitary measures applied four years after infection detection: 184.108.40.206. during that period, destroy volunteer potatoes and other host plants of the organism;
220.127.116.11. for the next three years after detection of the infection should not be planted Potato tubers, plants, sow the seeds, as well as to grow beets and other organism Syme niekaug to pārziemojuš of the potato field has at least two next vegetation period;
18.104.22.168. fourth year potatoes may be planted for food, feed or for processing, if at least two previous periods prior to planting of vegetation not found pārziemojuš potatoes and host plants. Fourth year shall be grubbed potatoes after harvesting checks according to point 3 of these rules;
22.214.171.124. current annual production of potatoes in crop rotation, seed potatoes may be planted for the production or other purposes (food, feed, processing) for potato production. These potatoes after harvesting, in accordance with paragraph 3 of this rule;
15.1.2. where a phytosanitary measures, applicable for five years after the infection detection: 126.96.36.199. during that period, destroy volunteer potatoes and other host plants of the organism;
188.8.131.52. the first four periods of vegetation in the install and maintain black fallow land, pasture land, which cuts the often intense grazing and low;
184.108.40.206. Fifth annual potato production, if at least two previous periods of vegetation not found pārziemojuš potatoes and host plants, potatoes may be planted seeds or other purposes (food, feed, processing) for potato production. These potatoes after harvesting, in accordance with paragraph 3 of this rule;
15.2. the other fields located in the infected area, which is not found in the pārziemojuš of potato plants and the organism which has tested the service, follow the crop rotation and following: 15.2.1 the first vegetation period after infection detection must not be planted Potato tubers, plants, to cause their seed or host plants of the organism. Ask the service to destroy the potatoes or permitted volunteer planting only certified seed potatoes for other purposes (such as food, feed, processing) for potato production, if it is destroyed, all pārziemojuš potatoes and organism and in the previous year have not grown beets;
15.2.2. second period of vegetation after the infection detection for use in food or seed production allowed to plant certified seed potatoes or potatoes, which have not found the body of the service, except that the place of production designated as contaminated;
15.2.3. in the third year of production of potatoes after detection of the infection for use in food or seed production allowed to plant certified seed potatoes or potatoes grown from certified seed potatoes;
15.2.4. in each of these regulations 15.2.1 and 15.2.2. vegetation period referred to in subparagraph service asks destroy volunteer potato and organism and monitor the implementation of the measure;
15.3. cover areas forbidden to plant Potato tubers and plants, as well as host plants of the organism, except for the production of potatoes used certified seed potatoes, or meristēm of sīkbumbuļ plants which have been checked in, as well as service is a complete replacement of the growing medium and cover the area and the cleaning and disinfection of equipment. 16. Service instructs the person under its supervision: 16.1. immediately after detection of the infection, as well as at the next vegetation period to clean up and disinfect the infected or suspect warehouses, agricultural machinery, vehicles and other objects, destroy or disinfected with infected or potentially infected potatoes on in-contact packaging. After disinfection, these objects are no longer considered infected or potentially infected;
16.2. immediately after the detection of infection and each 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 inventory, cover the areas of cleaning, as well as, if possible, disinfection;
16.3. cultivation sites on the infected area for at least three years after the infection detection: 16.3.1. seed potatoes to harvest, store and move separately from other purposes (food, feed, processing) for potatoes;
16.3.2. do all with potato production-related machinery, storage and cleaning of the area and, if possible, disinfection. 17. in order to determine the possible spread of the organism and the original provenance of the infection, the service check: 17.1. Potato tubers and plants associated with contaminated batches of potatoes;
17.2. the farm, located in the vicinity of the holding where the detected infected potatoes. 18. If the service body finds the original material, the Department carried out original material, random seed, basic or higher generation seed potato inspection, if their origin associated with infected seed potato lot and sent samples for laboratory analysis. 19. Service: 19.1. the decision shall specify the deadline of phytosanitary measures;
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 or potentially contaminated, after harvest, take the potato;
19.4. monitoring farms, warehouses and to move the potato tuber and rifts, and the wound holding agricultural technique used on a contractual basis, together with the holding on which it was found infected potatoes;
19.5. a decision shall be accompanied by a map of the infected area. 20. the infected area at least three the next vegetation period after infection detection service this provision referred to in paragraph 3. 21. 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, then the service may forcibly destroyed potato plantations chemical or mechanical means. 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 service 22, shall immediately inform the European Commission and the Member States of the European Union whenever a laboratory analysis of a positive test result. The notification shall specify the following: 22.1 subject to plant-health control of plants and plant products circulating in the registry of parties registered in the registration number of the holding if the holding is located in the place of production, set to be infected;
22.2. the infected host plants shipment or lot attached to the phytosanitary certificate or plant passport number;
22.3. the infected seeds or other purposes (food processing, fodder) potatoes varieties and seeds category;
22.4. the rules referred to in paragraph 10 of the test results of extract, prepared the slide immunofluorescence test, infected Eggplant (Solanum melongen) material and pure cultures of the organism. 23. the service may apply phytosanitary measures of infected and potentially infected potato use that are not listed in point 14 of these rules. Before the application of such measures shall inform the services of the European Commission and the Member States of the European Union. 24. If there is a risk of contamination of potatoes imported from another European Union Member State or exported to another Member State of the European Union, and is approved for potato ring rot, light service shall immediately inform the Member State concerned, shall be submitted together with the notification of plant passports and delivery a copy of the document. The notification shall specify: 24.1. infected potato variety name of the lot;
24.2. the sending and recipient's name and address;
24.3. potato delivery date;
15.2. the potato lot size;
15.2. the manufacturer's or dealer's registration number. 25. the service shall inform the European Commission of any organism is confirmed by the statement. The notice shall provide: 25.1. in certain cases. The description shall mention the number of places of production, number of lots of potatoes and potato varieties. Specifies the category of the seed potatoes;
25.2. the description of the investigation. The description shall mention the date that contamination was confirmed, the infected batch size and traceability (the source of contamination and the potential spread of infection);
25.3. a description of the zone. The description shall mention the number of places of production, not designated as contaminated, but included in the zone. IV. Closing questions 26. Be declared unenforceable in the Cabinet of 26 July 2005, the provisions of no. 569 "eradication of potato ring rot and control arrangements" (Latvian journal, 2005, 124 no). 27. paragraph 5 of these regulations shall enter into force by 1 January 2009. Informative reference to European Union directives, the regulations include provisions resulting from: 1) Council of 4 October 1993 of Directive 93/85/EEC on the control of potato ring rot; 2) Commission of 12 June 2006, Directive No. 2006/56/EC amending the annex to Council Directive 93/85/EC on the control of potato ring rot. Prime Minister a. Halloween Minister of Agriculture m. Roze Ministry of Agriculture presented version of annex 1 of the Cabinet of Ministers of 29 May 2007 No. 365 of the rules determining the Organism in Potato tubers and plants: 1. sample preparation 1.1. Potato tubers: 1.1.1. standard sample is 200 tubers per test. More intensive sampling requires additional tests using this size sample. If the number of tubers in the sample is larger, you may not do the test results or they will be difficult to interpret. However, if it is not available in the quantity required, this procedure is also good for samples with less than 200 tubers. All the methods described further validated by testing the samples of 200 tubers; 1.1.2. additional pre-treatment prior to sample preparation. Wash the tubers. Use appropriate disinfectants (when PCR-test, chlorine compounds are used to clear the potential pathogen DNA) and detergents for each sample. Allow the tubers to dry. The washing is helpful (but not required) for samples with excess soil and if test or PCR test for the direct isolation; 1.1.3. with a clean disinfected scalpel or vegetable knife the skin at each remove the heel end of the tuber, exposing the vascular. Cut out a small piece of potato heel end, trying to cut as little as possible tuber flesh. Select any tubers that have potential body symptoms and test separately. If the heel end of the environment during the removal of ring rot organisms apparent symptoms, perform a Visual inspection of the tuber, tuber come at the heel end. All tubers, which are respectively the body symptoms, not less than two days stored at room temperature to happen then woodiness, burying the tubers cool (4-100 ° C) in quarantine until the end of testing. All tubers including those who have suspicious symptoms stores; 1.1.4. collect the heel ends the middle piece of new disposable containers which can be closed or sealed (in case containers are reused, They shall be carefully cleaned and disinfected with chlorine-containing compounds). The heel end recommended process immediately. If this is not possible, they no longer than 72 hours refrigerated for not longer than 24 hours at room temperature in the container store, adding a buffer solution. Drying the piece of potato, woodiness and growth of Saprophytes during storage may hinder the cause of potato ring rot bacteria; 1.1.5. the process the heel end wire pieces of tissue, using one of the following methods: 220.127.116.11. the heel end pieces or transfuse with sufficient amount of extraction buffer (approx. 40 ml) in accordance with the provisions of annex 3 and four hours to shake on the Rotary shaker (50 to 100 revolutions/minute) at a temperature lower than 240 C, or 16-24 hours if they are chilled; 18.104.22.168. the vascular pieces homogenizing mixer (Waring or ultra Thurax), with enough (approximately 40 ml) of extraction buffer (Appendix 3), or with a rubber mallet or appropriate grinding (e.g. Homex) crush in tightly sealed in disposable maceration bag (e.g. Stomacher or Bioreba sterilized in a sealed polyethylene bag 150 mm x 250 mm). If the samples are homogenized mixer, reciprocal infection risk is very high. Take precautions to avoid aerosol formation or spillage during the extraction process. Ensure that each look game used freshly sterilised Blender blades and bowls. If the PCR test, avoid DNS transfer to containers or grinding devices. PCR test carried out disposable bags Shredder or containers; 1.1.6. the top clear supernatant liquid layer. If excessively cloudy, clarify either by slow speed centrifugation (10 minutes 4-100 ° C, not more than 180 grams) or using vacuum filtration (40-100 m) and washing the filter with additional extraction solution (10 ml); 1.1.7. concentrate the bacterial fraction, 4-100 ° C 15 minutes centrifugation with 7000 grams (or 10 minutes with 10 000 grams), the top layer of the clear liquid, not uzduļķoj to discard the sediment; 1.1.8. precipitate 1.5 ml of PBS to suspend again. Use the mkl organism testing, 500 and 500 mkl Ralstonia solanacearum and 500 mkl reference needs. The reference aliquot and to the remaining test aliquot add sterile glycerol to final concentration of 10-25% (v/v), 500 mkl, confused and keep-16 to-240 ° C (weeks) or at-68 to-860 C (months). During testing the remaining sample store 4-100 c. Repeated freeze and thaw the extract is not recommended. If the extract is transported, it carries the aukstumkast 24-48 hours; 1.1.9. the body positive controls and samples are treated separately to avoid infection of the nose. This applies to IF slides and to all tests. 1.2. potato plants: 1.2.1 to detect latent organism populations it is advised to test composite samples. Procedure may be used for composite samples of up to 200 stem parts (surveys used a statistically representative of the target population of plant samples). With a clean disinfected knife or pruning shears 1-2 cm segment from the base of each stem, just above the soil level. Long time not disinfect stem segments with 70% methanol and immediately Blot dry with tissue paper; 1.2.2. Insert stem segments in a closed sterile container according to the following sampling procedures: 22.214.171.124. process the stem segments by one of the following methods: 126.96.36.199.1. circle the stem segments with sufficient quantity (approximately 40 ml) of extraction buffer and shake on the Rotary shaker (50 100 revolutions/minute) for four hours at a temperature below 240 C or 16 to 24 hours refrigerated; handle 188.8.131.52.2. immediately. Stem segments in a homogenizing in the extraction solution with strong maceration bag (e.g. Stomacher or Bioreba,), crushing them with a rubber mallet or appropriate grinding (e.g. Homex). If you cannot immediately process the stem segments refrigerated can be kept for up to 72 hours, but room temperature-up to 24 hours; 184.108.40.206. stand up for 15 minutes, then top the clear layer Depr; 220.127.116.11. bacterial fraction extract or concentrate is not usually necessary in addition clarified, but it can be done by filtration or centrifugation as described above; 18.104.22.168. divide the neat or concentrated sample extract into two equal parts. One part of the store 4-100 ° C throughout the test, the second part, if you need further testing, by 10-25% (v/v) sterile glycerol is added to keep the horse-16 to-240 ° C (weeks) or at-68 to-860 ° C (months); 2. IF test: 2.1 IF test is recommended to be used as the principal screening test; 2.2. when the IF test is used as the principal screening test and the IFtest is positive, the second screening test is used in PCR or FISHtest. When the IF test is used as the second screening test and the if reading is positive, to complete the analysis, according to the testing scheme carried out additional testing. If the principal screening test used in the IF test, using a Polyclonal Antibody. When the IF test with Polyclonal Antibody is positive results, in addition to the screening with a monoclonal antibody can be better specificity, but lower sensitivity. Use a reference strain of the organism antibodies. It is recommended to determine the titre of antibodies to each new batch. The titre is defined as the highest dilution at which optimum reaction occurs when testing a suspension, which is 105 per ml to 106 Homo-organism strain cell window, and in accordance with the manufacturer's instructions, using an appropriate fluorescein isothiocyanate (FITC) conjugate. The crude Polyclonal and monoclonal antibody IF titre of at least 1:2 000. During testing, the antibodies used at a working dilution (DA), which equal to the titres or close to it. Use validated antibodies. The test is carried out using freshly prepared sample extracts. If necessary, you can successfully perform the test using the extracts that stored-up to 68-860 ° C glycerol solution. Glycerin from the sample by adding Venu ml pellet buffer by repeated centrifugation for 15 minutes with 7 000 g and resuspension of the same quantity of pellet buffer. This is often not necessary, especially if the sample is fixed to the slide with the flame. Prepare separate positive control slides of the homologous strain or any other reference strain of the organism, followed by potato extract, as specified in annex 9 of these rules or of the buffer solution. If possible, on the same slide for a similar use of control samples of naturally infected tissue (maintained by lyophilisation or freezing-16 to-240 ° C). On the negative control use a portion of the sample extract that previously tested negative. Use Multiwell microscope slides with preferably 10 Windows with a diameter of at least six millimetres. Control materials tested as samples; 2.3. prepare the test slides by one of the following procedures: 2.3.1 extracts with relatively little starch sediment. The first box of the pipette, transfer the appliance (15 ul corresponds to the box with six mm in diameter-for larger Windows increase proportionally to the quantity) of the resuspended potato pellet 1/100, then a similar volume of undiluted Pellet (1/1) onto the remaining boxes in the row. The second row can be used as duplicate or for a second sample (Figure 1); Figure 1 preparation of the Test slide for detection antibody titre 2.3.2. other extracts. Prepare the resuspended pellet decimal dilutions (1/10 and 1/100) buffer. Line the box with a pipette to transfer reference (15 ul corresponds to the box with six mm in diameter-for larger Windows increase proportionally to the quantity) of pure extract and suspension of each solution. The second row can be used as duplicate or for a second sample (Figure 2); Figure 2 Test slide preparation 2.4. dry the droplets at ambient temperature or heat a 40-450 (C). The bacterial cells to the slide by heating (15 min 600 ° C), flaming, with 95% ethanol or according to instructions from the suppliers of the antibodies. If necessary, fixed slides until further testing can be stored frozen way dry Chamber concluded shortly as possible (not more than three months); 2.5. IF procedure: 2.5.1. preparing slides for testing: 22.214.171.124. prepare a twofold dilutions of antibody in IF buffer. The first well is 1/2 of the titre (T/2), 1/4 of the titre of transition (T/4), 1/2 of the titre (T/2), the titre (T) full and double the titre (2T). 126.96.36.199. prepare the test slides. Prepare the working dilution of the antibodies (DA) in IF buffer. The working dilution affects the specificity; 2.5.2. the slides are placed on moistened paper. Each test box full overlay with the dilution of antibodies. The quantity of antibodies, which drops each box is equal to the volume of the extract. In the absence of specific instructions from the suppliers of the antibodies, perform the following procedure: 188.8.131.52. incubate slides 30 minutes to wet the paper covered way at ambient temperature (18-250 C); 184.108.40.206. for each harvest drops from the slide and Rinse carefully with IF buffer. Wash for five minutes in IF buffer-Tween dipping and then for five minutes in IF buffer. To avoid aerosol or droplet transfer that may result causing contamination. Carefully remove excess moisture, easy to dry; 220.127.116.11. slides shall be placed on damp paper napkins. Cover the test Windows with the dilution of FITC conjugate used to determine the titre. Is the volume of conjugate applied on the box, is equal to the volume of antibody used; 18.104.22.168. incubate slides 30 minutes to wet the paper covered way at ambient temperature (18-250 C); 22.214.171.124. droplets of conjugate to shake off the slide. Rinse and wash as above. Wipe excess liquid; 126.96.36.199. pipette 5 on each window-mkl 10 0.1 M phosphate-buffered glycerol or a commercially antifading mountant promotes stability, color and apply a coverslip; 2.6. reading the IF test: 2.6.1. examine test slides on an epifluorescence microscope oil or water immersion at a magnification of 500 to 1000, with filters suitable for working with FITC. The boxes look across two diameters at right angles and around the perimeter. With respect to samples show a small quantity of cells, consider at least 40 microscope fields. First check the positive control slide. The cells must be bright fluorescent and completely stained at the determined antibody titre or working solution. If test is repeated if the staining is aberrant. 2.6.2. the test Windows of the test slides for bright Fluorescing cells with characteristic morphology of the organism. Fluorescencesintensitāt must be the same for the positive control strain at the same antibody dilution or more intense. Cells with incomplete staining or with weak fluorescence is not taken into account. Repeat the test if any contamination is suspected. This may be the case if all the lots in question slides show positive cells due to the contamination of buffer or if positive cells are found on the coverslip (outside of the slide Windows); 2.6.3. There are several problems inherent to the immunofluorescence test. Not the typical morphology of the fluorescent background populations of cells and cross reacting saprophytic bacteria with size and morphology similar to organisms likely to occur in potato heel and notice of potato stem segment; 2.6.4. consider only Fluorescing cells with size and morphology, which is a typical antibody titre or working dilution; 2.7. IF test reading interpretation: 2.7.1. If bright Fluorescing cells with characteristic morphology are found, to determine the characteristic of the average number of cell microscope field and calculate the number of typical cells per ml of resuspended Pellet. IF the test result is considered those samples with no less than 5 × 103 typical cells per ml of resuspended Pellet. Sample is considered potentially infected and needs further testing; 2.7.2. IF the test result is considered negative for samples with less than 5 x 10 typical or non-typical cells per ml of resuspended pellet and the sample is considered negative. The test is not carried out in the future. 3. FISH test: 3.1 when the FISH test is used as the first screening test and the FISH test is positive, as a second compulsory screening test use the IFtest. When the FISH test is used as the second screening test and the if reading is positive, to determine the final diagnosis, conduct further testing. Special use validated c. m. subsp. sepedonicus oligo. The mandatory requirement is that the previous jie, this method tests to ensure reproducible detection of 103 to 104 cells of the organism-one mililir, which added to sample extracts which previously tested negative. The following procedure should preferably be performed on freshly prepared sample extract but can be successfully implemented, using the sample extract stored in glycerol solution-16 to-240 to 68-860 C or-c. As negative controls, use part of the sample extract that previously tested negative for the presence of the organism. As positive controls prepare suspensions containing 105 to 106 cells per ml of the organism (e.g. NCPPB 4053 or PD 406 strain strain) 0.01 M phosphate buffer (PB), using a 3-5 day culture. Prepare separate homologous body or any other reference strain of the positive control slides using potato suspension. With FITC labelled will use of oligo hybridisation process control because its stained all the will in the sample. Test control material like sample; 3.2. potato extract fixation: 3.2.1. Protocol is based on a Wulling et al. (1998); 3.2.2. prepare fixative solution; 3.2.3. transfer 100 mkl each sample extract into an Eppendorf tube and centrifuge for eight minutes with 7 000 grams; 3.2.4. remove the supernatant and dissolve the pellet in 500 mkl of fixative prepared at least 24 hours in advance. Mix and 40 C temperature until the next day. As an alternative, you can use a fixative 96% ethanol. To use it, dissolve the Pellet, provided 50 0, 01 m PB mkl and 50% ethanol 96 mkl. Mix and incubate for 40 ° C, 30-60 minutes; 3.2.5. centrifuge for eight minutes with 7 000 grams, remove the supernatant and resuspend the pellet in 0, 01 m PB mkl 75; 3.2.6. transfer the suspension on 16 locking mkl clean heavily used slide (Figure 3); Figure 3 diagram of each FISH slide slide is spotted two different samples, and undiluted 1:100 dilution preparation (0, 01 m Pb), use the mkl 10. The remaining solution (49 mkl), adding 1 v. 96% ethanol, can be stored at-20 ° c. Year, when the FISH you repeat the test, remove the ethanol by centrifugation and add the same amount of 0, 01 m PB (mix by vortexing); 3.2.7. allow slides to air dry (or the freeze-dried desiccator 37 ° C) and fix with the flame. At this stage, the procedure may be interrupted, and hybridisation may continue the next day. The slides are stored at room temperature in a dry room that does not have the dust; 3.3. preparation and hybridization hybridization: 3.3.1 prepare a lysozyme solution containing 10 mg lysozyme (Sigma L-6876) 10 ml of buffer solution (100 mM TRIS-HCl, pH 8.0, ADT, 59 mM). This solution can be stored, but may be frozen and thawed only once. Each sample shall be covered with about 50 mkl lysozyme solution and kept at room temperature for 10 minutes. Then the slides once demineralised water and dry with filter paper. Alternatively, instead of lysozyme into each well 50 40-400 mcg per ml mkl-1 K proteniāz-buffer (20 mM TRIS-HCl, 2 mM CaCl2, pH 7.4) and 370 ° C incubate for 30 minutes; 3.3.2. the cells dehydrate sequentially on one minute dipping 50%, 80% and 96% ethanol. Dry the slide rack slides; 3.3.3. prepare a moist incubation chamber lined the bottom of a box with tissue or filter paper soaked once hybridisation solution. Prepare the box for at least 10 minutes for the hybridisation oven at 550 ºc; 3.3.4. to prepare the hybridisation solution, providing for each slide, and 45 mkl 5 minutes to hold 550 C; 3.3.5. place slides on hotplates 450 ° C and add 10 mkl hybridisation solution all four wells on the slide; 3.3.6. each slide to impose two coverslip (24 x 24 mm), displacing the air. Hybridization of the slides until the next day to place the darkness warmed humidity chamber above 550 ° C in the oven; 3.3.7. prepare three beakers containing one litre of ultra pure water, one liter of 1 x hybmix (334 ml 3 x hybmix and 666 ml ultra pure water) and 1 l 1/2 x hybmix (167 ml 3 x hybmix and 833 ml ultra pure water). They prepare for 550 ° C in a water bath; 3.3.8. of slides remove coverslip and place the slides in the rack; 3.3.9. Rinse the excess quantity of the sample, 15 minutes the beaker with 1 x hybmix of 550 ° C; 3.3.10. Insert slide rack 1/2 hybmix washing solution and incubate for a further 15 minutes; 3.3.11. briefly dip the slides in upw and place on absorbent paper. Dry the excess moisture, surface covered with filter paper carefully. In each box, add 5-10 mkl of anti-fading mountant solution (e.g. Vectashield, vecta laboratories, Canada, USA or equivalent) cover the whole slide a large coverslip (24 x 60 mm); 3.4. Reading the FISH test: 3.4.1. with an epifluorescence microscope slides immediately immersion oil look at 630 or 1000 x increase in you. With a filter suitable for fluorescein isothiocyanate (FITC), will the cells (including prominent in gramnegatīv cells) in the sample stained fluorescent green. Using a filter for tetramethylrhodamine-5-isothiocyanate, Cy3-stained cells of the body's cells appear fluorescent red. Compare cell morphology with the positive controls, morphology. The cells must be bright fluorescent and completely stained. The fish test is aatkārt, if the staining is aberrant. The boxes look across two diameters at right angles and around the perimeter. With respect to samples show a small quantity of cells, at least 40 microscope fields; 3.4.2. in the test Windows of the test slides for bright Fluorescing cells with characteristic morphology of the organism. The fluorescence intensity must be equivalent to the positive control strain, or more intense. Cells with incomplete staining or with weak at gu fluorescence is not taken into account; 3.4.3. If any contamination is suspected, the test is repeated. This may be the case if all the lots in question slides show positive cells due to the contamination of buffer or if positive cells are found on the coverslip (outside of the slide Windows); 3.4.4. There are several problems inherent to the FISH test. In potato heel end core and stem segment pellets may occur with background populations of Fluorescing cells with atypical morphology and cross reacting saprophytic bacteria with size and morphology of the organism, but it happens a lot less often than do the IF test; 3.4.5. consider only Fluorescing cells with typical size and morphology. 3.4.6. interpretation of the FISH test: 188.8.131.52. valid FISH test results are obtained, if at all in the positive control preparations when using the FITC filter detects a bright green fluorescent cells with body size and morphology typical of and observed using the rhodamine filter finds red fluorescent cells, and they are not found in any of the negative controls. If bright Fluorescing cells with characteristic morphology are found, to determine the characteristic of the average number of cell microscope field and calculate the number of typical cells per ml of resuspended Pellet (annex 4). Samples with no less than 5 × 103 typical cells per ml of resuspended pellet are considered potentially contaminated. Need further testing. Samples with less than 5 x 10 typical cells per millilitre of resuspended pellet are considered negative; 184.108.40.206. FISH test is negative if the observed using the rhodamine filter are not bright red fluorescent cells with body size and morphology typical of, provided that typical bright red fluorescent cells are observed in the positive control preparations when using the rhodamine filter; 4. PCR test: 4.1 if the PCR test is used as the principal screening test and found to be positive, the IF test must be performed as a second compulsory screening test. When the PCR test is used as the second screening test and the if reading is positive, to determine the final diagnosis, further testing according to the sample testing scheme. Use this method as the principal screening test is only recommended in cases where experience was gained in its use. Preliminary testing with this method should permit reproducible 103 to 104 cells of organisms per millilitre of the discovery added to sample extracts which previously tested negative. Optimisation experiments may be required for all laboratories to achieve maximum sensitivity and specificity. Use of validated PCR reagents and protocols. It is recommended that you choose a method of internal control. Take appropriate precautions to avoid contamination of the sample with target DNA. The PCR test is carried out by experienced technicians, in dedicated molecular biology laboratories, in order to reduce the possibility of contamination of the sample with target DNA. Negative controls (for DNA extraction and PCR) is always used as the final model of the procedure to determine whether the DNA transfer has taken place; 4.2. PCR test includes the following negative samples: 4.2.1 the sample extract that previously tested negative for the presence of the organism; 4.2.2. sample buffer solution used for extracting the bacterium and the DNA from the sample; 4.2.3. PCR reaction mix; 4.3. following positive controls: 4.3.1. re-suspended extract parts, which added to the body; 4.3.2. from virulent isolates obtained water suspension with 106 body cells per millilitre (NCPPB 2140 or NCPPB example 4053); 4.3.3. If possible, also use DNA extracted from positive control samples in the PCR test. To avoid potential contamination prepare positive controls spatial separation separately from samples. Sample extracts from cleans the soil as possible. Therefore, when used in a PCR Protocol, it is recommended that you extract the votes of sagat washed potatoes; 4.4. Dna purification methods: 4.4.1. use positive and negative control samples as described above. Prepare control material like sample; 4.4.2. complex sample substrates, the target DNA is available for the treatment of different methods to separate the PCR inhibitors, prevent other enzymatic reactions and concentrating target DNA in the sample extract; 4.4.3. for use with the validated PCR method is optimized for the following method: 220.127.116.11. Pastrik (2000). 18.104.22.168.1 transfer method: 220 mkl lizēšan buffer (100 mM NaCl, 10 mM TRIS-HCl [pH 8,0], 1 mM EDTA [pH 8,0]) 1.5 ml into an Eppendorf tube; 22.214.171.124.2. Add 100 mkl sample extract and place the 10 min in a heating block or water bath at 950 ° C; 126.96.36.199.3. the test tube for 5 minutes to hold ice; 188.8.131.52.4. add 80 mkl lysozyme solution (50 mg lysozyme per ml in 10 mM TRIS HCl, pH 8.0) and 30 min hold 370 ° C; 184.108.40.206.5. Add 220 mkl Easy DNA ® solution A (Invitrogen), mix well and hold 650 ° C for 30 minutes; 220.127.116.11.6. Add 100 mkl Easy DNA ® solution B (Invitrogen), mix by vortexing carefully until the sample is uniformly viscous; 18.104.22.168.7. Add 500 mkl of chloroform and stir until the viscosity decreases and the mixture becomes homogeneous; 22.214.171.124.8. centrifuge for 20 min 40 ° C at 15 000 g to separate phases and form the interphase; 126.96.36.199.9. transfer the upper phase into pure microvial; 188.8.131.52.10. Add one millilitre of the 100% ethanol (-200 C), briefly stirred vigorously and stand for 10 minutes on the ice; 184.108.40.206.11. centrifuge for 20 min 40 ° C at 15 000 g and remove ethanol from pellet; 220.127.116.11.12. Add 500 mkl 80% ethanol (-200 C) and mix by inverting the tube; 18.104.22.168.13. centrifuge for 10 minutes at 40 ° C at 15 000 g, save the pellet and remove ethanol. 22.214.171.124.14. to allow the extract to dry at room temperature or DNS vakuumžāvētāj; 126.96.36.199.15. resuspend the pellet in 100 mkl sterile ultra pure water and leave at room temperature for at least 20 minutes; 188.8.131.52.16. to use PCR to store-200 ° C; 184.108.40.206.17. spin down any white precipitate by centrifugation and use 5 PCR mkl supernatant containing DNA; 220.127.116.11. other DNA extraction methods (e.g. Qiagen DNeasy plant, Kit) can be used, provided that it is demonstrated effectiveness equivalent to DNA from control samples in the treatment that one millilitre of 103 to 104 pathogen bacteria content. 4.4.4. PCR: PCR 18.104.22.168. prepare test and control samples in accordance with a validated Protocol. Prepare one obtained from the sample's DNA extract decimal (1:10 in upw); 22.214.171.124. According to published protocol to prepare the appropriate PCR reaction mix in a contamination-free environment. Validated PCR Protocol is a multiplex reaction that also incorporates an internal PCR control; 126.96.36.199. Add 5 mkl DNA extract per 25 sterile PCR PCR reagent mkl tubes; 188.8.131.52. prepare a negative control sample containing only PCR reaction mix and add the sample in the same kind of sterile water, which was used in the PCR mix; 184.108.40.206. Insert tubes in the same either, used in the previous tests, and implement the optimized PCR program in accordance with the provisions of annex 5; 4.4.5. The PCR product analysis: 220.127.116.11. split the PCR Amplicon with agarose gel electrophoresis. You do this with 5-8 V/cm, taking at least 12 mkl DNS propagation reaction mix of each sample mixed with 3 sample loading buffer mkl (annex 5) 2.0% (w/v) agarose gel in three-to-actet EDTA (Tae) buffer. Use the appropriate DNA marker, such as "100 bp ladder"; 18.104.22.168. to highlight the DNS zone, 30-45 mins its colors with ethidium bromide (0.5 mg/l), taking appropriate precautions for handling this mutagen a substance; 22.214.171.124. PCR products size corresponds to light coloured gel, with shortwave UV transiluminator (for example, X = 302 nm), and to document the scene; 126.96.36.199. all newly detected cases verify authenticity of the PCR Amplicon by performing restriction enzyme analysis of the remaining DNS propagation model, the optimum temperature for optimal time by incubating with an appropriate enzyme and buffer. Identify the sections that cracked agarose gel electrophoresis methods specified above, and fade with the passage of UV transiluminator and restriction inherent with enzymes and staining with ethidium bromide. Compare the positive testimony before the break and after; 4.4.6. PCR test result interpretation: 188.8.131.52. when the PCR test is negative if the organismamraksturīg PCR Amplicon of expected size in that pattern is not found, but it finds in all positive kontroltesto in the case of multiplex PCR with plant specific internal control primers: a second PCR-product of expected size must be amplified with the sample; 184.108.40.206. PCR test is positive if the organism is detected specific PCR Amplicon of expected size with characteristic and restriction (if necessary), provided that it is not derived from any of the negative control samples. The positive results of the safe the approval may also be obtained by repeating the test with a second set of PCR primers. The PCR may be suspected if the expected Amplicon is obtained from the positive control sample containing c. m. subsp. sepedonicus in water, but the positive controls with c. m. subsp. sepedonicus in potato extract produced negative results. Multiplex PCR Protocols with internal PCR controls of the reaction to the fact that neither of the two amplicons are obtained. Contamination may be suspected if the expected Amplicon is obtained from one or more of the negative control. 5. bioassay test: 5.1 the previous guide testing this method ensures reproducible detection of 103 to 104 organisms of colony-forming units per ml added to sample extracts which previously tested negative. The highest detection sensitivity expected using freshly prepared sample extract and to ensure optimal conditions for development. However, this method can be used successfully also extracts that stored-up to 68-860 ° C glycerol solution. Some Eggplant varieties are excellent selective culture medium for the development of the organism, even if there are no symptoms, as well as providing excellent host for confirmatory test. To reduce false negative test result the risk of developing conditions must be optimal, in accordance with the provisions in annex 7 of the growing conditions. 5.2. split the resuspended pellet in the rest part of the eggplants, using one of the methods described below. Use only plants 2-3 leaf stage of development until completely evolved third real page. To ensure that the resuspended Pellet is used completely and following inoculation process to be effective, the procedure below to complete the sample, comprising 15-25 eggplants; 5.3. do not water eggplants for two days before the start of inoculation to reduce turgor pressure; 5.4. slit inoculation: 5.4.1. holding the plant between two fingers, on the stem between the cotyledons and the first leaf pipette a drop of resuspended Pellet (approximately 5 to 10 mkl); 5.4.2. using a sterile scalpel, about 1.0 cm long and approximately 2/3 of the stem thickness deep, diagonal cut, starting the cut from the point where the pellet drop; 5.4.3. seal the cut with sterile Vaseline from a syringe; 5.5. syringe inoculation: 5.5.1. inoculate the Eggplant stems immediately above the cotyledons using a syringe fitted with a hypodermic needle (not less than 23 g). Distribute the pellet between the eggplants; 5.5.2. the positive control inoculate five plants for a water suspension of one millilitre contains 105 to 106 known organism in culture cells, and, if possible, with naturally infected tuber tissue, using the same inoculation method; 5.5.3. negative control five plants inoculated with sterile pellet using same inoculation method; 5.5.4. the quarantine conditions the plants 18-240 ° C at four weeks. Incubate plants with sufficient light, suitable humidity (70-80%) and released to prevent water logging or wilting through water deficiency. The body's cells are killed at temperatures above 300 C, but optimum temperature is 210 C development. to prevent contamination, positive control and negative control sample incubate on individual greenhouse shelves or breeding Chamber or, if there is not enough space, strictly to separate the treatment material. If the plants are different samples incubated close together, separated with the appropriate screens. When fertilising, watering, inspecting and performing other actions, is careful to avoid cross-contamination. It is very important to glasshouses pests because they can transfer bacteria from one plant to another. 5.5.5. at the start of the week the symptoms on a regular basis. Count how many plants are characteristic symptoms. Organism causes leaf wilting in eggplants which may commence as a ļenganum page or between the edges of the leaves stripped. Wilted tissue may initially have a dark green color or spots, but before death they become paler. Wilted tissue between the veins of the leaves often appear as greasy and as if filled with water. Dead skin tissue is often bright yellow line. Plants can not fully died, the longer is the period when illness develops, the greater the chance of survival of plants. Plants can withstand infection. Against Mike roorganism of susceptible young eggplants are much more sensitive to small body populations than older plants, hence the need to use plant leaves 3 stage or just before it. Wilts may also be caused by other bacteria or fungal populations that may be in the tuber tissue Pellet. They can be of Ralstonia solanacearum, Erwinia carotovora, subsp. carotovora and e. carotovora subsp. atroseptica, Erwinia chry-santhem, Phoma exigua var. foveat, as well as large populations of saprophytic bacteria. In particular Erwinia Chrysanthemi can cause leaf symptoms and wilt, which is very similar to c. m. sepedonicus symptoms. The only difference is blackening trunk of Erwinia Chrysanthemi infections. Other Wilts can be distinguished from those caused by the organism, since whole leaves or whole plants wilt rapidly. You can also prepare a Gram stain-this test is different from the body of Erwinia spp.; 5.5.6. when eggplants are observed symptom, re-accreditation of insulation, using savītušo in leaf tissue and stem tissue. Surface disinfect the Eggplant leaves and stems by wiping with 70% ethanol. Make Eggplant juice IF test or PCR and isolate the appropriate (selective) medium. You can also prepare a Gram stain. Identify pure cultures of the organism and may confirm the pathogenicity; 5.5.7. under certain circumstances, in particular where growing conditions are not optimal, the body can exist as a latent infection within eggplants even after incubation for four weeks. If the symptoms are not observed after four weeks, the IF or PCR test carried out United sample, consisting of one centimeter long stem sections of each test plant taken above the inoculation site. If the test result is positive, the repeated isolation of suitable (selective) media. Identify pure cultures of the organism and may confirm the pathogenicity as defined above; 5.6. the interpretation of the bioassay test: 5.6.1. valid bioassay test results are obtained, if the positive control plants finds typical symptoms of these plants, bacteria can be isolated again, and negative symptoms in kontroltesto not found. 5.6.2. bioassay test is negative if test plants are not infected with the organism, the organism is detected in positive kontroltesto; 5.6.3. bioassay test is positive if the plants are infected with the organism. 6. isolation of the Organism: 6.1 the diagnosis can be confirmed if the organism has been isolated, identified and then confirmed with the pathogenicity test. Although the organism is a fastidious organism, it can extract from symptomatic tissue. However, you can suppress saprophytic bacteria that quickly develops, and so directly from the tuber or stem tissue Pellet is complex. With a selective medium and corresponding resuspended potato heel end cores or stems of the Pellet is possible direct isolation of the organism. The isolation is carried out all the Potato tubers or stem segments and aubergines, which symptoms are not present, but for which the IF or PCR test results, using pooled sample was positive. Positive control of the use of decimal dilutions of the suspension, which is 106 colony-forming units per millilitre of the organism (e.g. NCPPB 4053 or PD 406,). To avoid possible infection, his positive test in fully separated from the samples for testing. Each newly prepared batch of selective medium before testing the sample check its conformity with the development of the pathogen. Test control material like sample; 6.2. selective plating: 6.2.1. using 100 mkl resuspended potato pellet or Eggplant SAP make 10-fold, dilution of the pellet buffer (Appendix 3); 6.2.2. isolation from undiluted potato pellet usually fails because the CMS is very sensitive to growth and competition by Saprophytes. Whereas bacteria in infected tissues are usually found in large populations, can dissolve the Saprophytes retaining pathogens. Therefore, it is recommended that each sample 100 mkl from 1/100 to 1/10 000 dilution to MTNA medium or NCP-88 medium (using Petri dishes with a diameter of 90 mm, other container sizes adjusted accordingly) by using the rod (glass rod) and by a method that provides for the alignment of the sample on the plate. An alternative is 100 mkl initial potato pellet onto the first align the agar plate with a rod, and then to the existing balance of the rod to wipe along the second agar plate; Finally the same repeat along the third plate, thus prepare a dilution of the rod; 6.2.3. incubate plates in the dark 21-230 ° C; 6.2.4. the initial examination of the wide between them compared with the control boards, after three days of total body-like colonies, future census taking in five, seven, and finally after 10 days; 6.3. the suspicious treatment of colonies: 6.3.1. to take the body to the culture YGM-like colony subculturing in the future to make Eggplant inoculation of plants or identification; This must be done before the plates are overgrown, after 3-5 days; 6.3.2. uzsē the body in similar colonies on one of the following feeds: 220.127.116.11. nutrient dextrose agar (for use only for en route); 18.104.22.168. yeast peptone glucose agar; 22.214.171.124. nutrient yeast extract mineral salts agar; 126.96.36.199. incubate at 21-240 ° C, up to 10 days. The body is slow-growing organism, usually 10 days make up a tiny little (how the spine cusp), cream, domed colonies; 6.4. The tirage liqueur en route. Growth rates will increase after the inoculation. Typical colonies are creamy-white or ivory, yellow, sometimes very noap, smooth, raised, convex-domed, mucoid-fluidal, with entire edges and usually from 1 to 3 mm in diameter. The usual painting of a Gram can help you choose the colony further testing; 6.5. identify possible culture and make a pathogenicity test. 7. identification: 7.1 identify possible body isolate pure cultures using at least two of the following tests based on different biological principles. Where appropriate, to include in the text of the reference strains for each test performed; 7.2. the growing medium and enzymatic identification tests: 7.2.1 determine the following phenotypic traits that constantly or not detectable in the body in accordance with the methods and Lelli Stead (1987), Klement et.al. (1990), Schaad (2001), anonymous aut. (1987); 7.2.2. all feeds 210 ° C and incubate for review after six days. If growth occurs, they incubate for up to 20 days. 7.2.3. test organism shall be included in all tests. Nutrients and enzymatic identification tests should be carried out in relation to the inoculum from nutrient agar subcultures. Morphological comparisons made using crops grown on nutrient dextrose agar: tests results Expected oxidation/fermentation test (O/F) Inert or weakly oxidative oxidase activity increase of 370 C of urease aesculin hydrolysis + develop starch hydrolysis-or poorly expressed tolerance against 7% NaCl indole test of catalase activity + H2S development-citrate consumption gelatin liquefaction-acid from glycerol acid from lactose-or weak acid from the acid from rhamnose salicīn Gram stain + 7.3. IF test : 7.3.1. prepare a suspension IF buffer that contains approximately 106 cells per ml; 7.3.2 prepare the appropriate dilution of antiserum double series; 7.3.3. IF procedure; 7.3.4. IF test is positive if the IF titre of the culture is the same as the titre of the positive control; 7.4. PCR test: 7.4.1. prepare a suspension in upw, approximately 106 cells ml; 188.8.131.52 ul of cell suspension in closed tubes in either the or boiling water bath for four minutes to heat 1000 c. If necessary, just add the NaOH prepared a final concentration of 0.05 M can help the cell lizēšan. The samples can be stored-up to 16-240 ° C until they are needed; 7.4.3. take appropriate PCR procedures to replicate the organism-specific Amplicon; 7.4.4. the identification of the organism is positive if the PCR amplicons are the same restriction fragment length polymorphisms and Amplicon are equal in size to the positive control strain. 7.5. FISH test: 7.5.1. prepare a suspension in upw, approximately 106 cells ml 7.5.2. make a FISH; 7.5.3. A positive FISH test is achieved if the culture and the positive control reaction is the same. 7.6. fatty acid profiling (FAP): test 7.6.1. grow the culture on triptāz's soy agar (Oxoid) for 72 hours at 210 ° C (± 10 (C)); 7.6.2. apply an appropriate FAP procedure (Janse, 1991; Stead, 1992); 7.6.3. A positive FAP test is achieved if the possible cultural profile is equal to the positive control profile. Fatty acids are 15:1 anteiso A, 15:0 ISO, 15:0 anteiso, 16:0 ISO, 16:0 and 18:0 anteiso is typical of c. m. sepedonicus. Genera belonging to other bacteria, such as curtobacterium, Arthrobacter and Micrococcus also have some of these fatty acids but 15:1 anteiso A is a rare acid in these bacteria; It is in all Clavibacter spp. ranging from 1% to 5%. C. m. sepedonicus bacteria this amount is usually around 5%; 7.7. BOX-PCR: 7.7.1. prepare a suspension in upw, approximately 106 cells ml; 7.7.2. to carry out the test in accordance with the procedure (Smith et al., 2001). 8. Approval test: 8.1. pathogenicity test performed to definitively confirmed the diagnosis and confirm the virulence of cultures identified as organism; 8.2. the test isolates from three days to prepare an inoculum of plating, approximately 106 cells ml, and prepare the positive control strain of the organism; 8.3. inoculate 5 to 10 Eggplant stems of young stems of three leaf stage; 8.4. incubate at 18-240 ° C with sufficient light and high relative humidity with appropriate watering to avoid waterlogging or drought stress. The characteristic of a pure wilt observed within 2 weeks, but plants with no signs of infection after that time, continue to grow to 3-week period to achieve the temperatures, which promote the development of the eggplants, but no higher than 250 C. If after 3 weeks the symptoms are not present, the culture cannot be confirmed as being a pathogenic form of the organism; 8.5. symptomatic plants to isolate part of a tree trunk cut 2 cm above the inoculation site. Crush and suspend a small quantity of sterile distilled water or 50 mM phosphate buffer. ISO from the suspension applied to me or strapped to the dilution of MTN or YPG, 3-5 days at 21-230 ° C and observe the body's typical colony formation.
Minister of agriculture m. Rose proposed the Ministry of agriculture in annex 2 versions of the Cabinet of Ministers of 29 May 2007 regulations no 365 feeds body isolation and cultivation 1. General growth media nutrient agar (NA) nutrient agar (difco) 23.0 g distilled water 1.0 l 1.1. dissolve ingredients and autoclave 15 minutes at 1210 ° C; 1.2. the nutrient dextrose agar (NDA); 1.3. nutrient agar (difco bacto) containing 1% d(+) glucose (monohydrate). 20 min sterilization autoclave 1150 ° C; 1.4. yeast peptone glucose agar (YPG): yeast extract (difco) 5.0 g bacto-peptone (difco) 5.0 g D (+)-glucose (monohydrate) 10.0 g Baktoagar (difco) 15.0 g distilled water 1.0 l 1.5. dissolve ingredients and autoclave for 15 min at 1210 C temperature. 1.6. the yeast extract mineral salts medium (YGM); The Bakt yeast extract (difco) 2.0 g D (+) glucose (monohydrate) 2.5 g K2HPO4 0.25 g KH2PO4 0.25 g 0.1 g MnSO4 MgSO 4.7 H2O. H2O 0.015 g NaCl 0.05 FeSO 4.7 H2O 0.005 Baktoagar (difco) 18 g distilled water 1.0 l 1.7. Dissolve ingredients and sterilize 0.5 l went through a 20 min in autoclave c. 1150. 2. Validated selective growth media MTN medium unless otherwise specified, all the feeds components are from BDH: yeast extract (difco) 2.0 2.5 g K2HPO4 0.25 g mannitol 0.25 g NaCl 0.05 g H2PO4 g 0.1 g MnSO4 MgSO 4.7 H2O. H2O 0.015 FeSO 4.7 H2O0 0.005 Agar (Oxoid No. 1) 16.0 g distilled water 1.0 l 2.1. dissolve ingredients to balance pH to 7.2. After autoclaving (1210 C for 15 min) and cooled to 500 C, add the antibiotics: trimethoprim 0.06 g, nalidixic acid 0.002 g, amphotericin B 0.01 g; 2.2. standard solution of antibiotics: trimethoprim (Sigma) and nalidiksīnskāb (Sigma) (both 5 mg/ml), 96% methanol, amf-tericīn B (Sigma) (1 mg/ml) in dimethyl sulfoxide. Stock solutions are filter-sterilized; Note: the determination of the expiry period is 3 months. After the addition of the antibiotics the validity period is 1 month if stored refrigerated. NCP-88 medium nutrient agar (difco) 23 g yeast extract (difco) 2 g D-mannitol K2HPO4 5 g 2 g KH2PO4 0.25 0.5 g H2O MgSO 4.7 g distilled water 1.0 l 2.3. dissolve ingredients, adjust pH to 7.2. After autoclaving and cooling to 500 (C) add the following antibiotics: polymyxin B sulphate (Sigma) 0.003 g, nalidixic acid (Sigma) 0.008 g, cycloheximide (Sigma) 0.2 g; 2.4. dissolve antibiotics in the standard as follows: nalidixic acid 0.01 M NaOH, cycloheximide in 50% ethanol, polymyxin B sulphate in distilled water. Stock solutions are filter-sterilized. Note: the determination of the validity is three months. After the addition of the antibiotics the period of validity is one month if stored refrigerated.
Minister of agriculture m. Roze Ministry of Agriculture presented the annex 3 of the Cabinet of Ministers of 29 May 2007 regulations no 365 buffer test procedures 1.-Sterile buffers can be stored for up to one year; 1.1. extraction procedure buffer. Extraction buffer (50 mM phosphate buffer, pH 7.0). This buffer is used for the extraction of bacteria from plant tissues by homogenisation or shaking method: Na2HPO4 4.26 g (without water) KH2PO4 2.72 g distilled water 1.00 l 1.2. dissolve ingredients, check pH and autoclave for 15 min at 1210 C temperature. Additional components can be used as follows: the target quantity (per l) Lubrol flakes deflocculant (*) 0.5 g DC silicone antifoam agent anti-foaming agent (*) 1.0 ml Tetrasodium pyrophosphate antioxidant 1.0 g polyvinylpyrrolidone-40000 (PVP-40) PCR inhibitors saistīšn 50 g (*) for use with homogenisation extraction method. 1.3. Pellet buffer (10 mM phosphate buffer, pH 7,2). This buffer is used for dilution of resuspended and extract of potato tuber heel end pieces, which concentrated with centrifugation method. H2O 2.7 g NaH2PO Na2HPO 4.12 H2O 0.4 g 4.2 distilled water 1.00 l dissolve ingredients, check pH and autoclave for 15 min at 1210 C temperature. 2. IF test buffer: 2.1 IF buffer (10 mM phosphate buffered saline sodium chloride solution (PBS), pH 7,2). This buffer is used for dilution of antibodies: H2O 2.7 g Na2HPO 4.12 H2O 0.4 g NaCl NaH2PO 4.2 8.0 g distilled water 1.0 l dissolve ingredients, check pH and autoclave for 15 min at 1210 C temperature. 2.2. IF-Tween. This buffer is used for washing the slides. Add 0.1% Tween 20 to the IF buffer. 2.3. Phosphate buffered glycerol pH 7.6., this buffer is used as a mountant fluid deposit boxes IF slides to enhance fluorescence.
H2O 3.2 g NaH2PO Na2HPO 4.12 4.2 H2O 0.15 g glycerol 50 ml distilled water 100 ml fading mountant fluid persistent are sold ready e.g. Vectashield ® (Vector laboratories) or Citifluor ® (Leica).
Minister of agriculture m. Roze Ministry of Agriculture presented the annex 4 of the Cabinet of Ministers of 29 May 2007 regulations no 365 contamination level detection with IF and FISH tests 1. Count the mean number of typical fluorescent cells per field (c).
2. Calculate the number of typical fluorescent cells per microscope box (C).
(C) = c x S/s, where S = Multiwell microscope slide one surface area and s = objective Visual field area.
s = 4G2/K2/Tri2/where i = field coefficient (depending upon ocular type can be anywhere from 8 to 24), K = tube coefficient (1 or 1,25), G = magnification of objective (100 x, 40 x, etc.).
3. calculate the number of typical fluorescent cells per ml of resuspended Pellet (N).
N = C × 1 000/y, where y = F x of resuspended pellet quantity in each box, and F = of resuspended pellet dilution factor. Minister of agriculture m. Roze Ministry of Agriculture presented versions of annex 5 of the Cabinet of Ministers of 29 May 2007 regulations no validated PCR Protocol and 365 reagents 1. reproducible detection of previous test sensitivity of at least 103 to 104 c. m. sepedonicus cells per ml of sample extract. Also, previous tests may show no false positive results for the selected bacterial strains 2. PCR Protocol of Mmkltipl with internal PCR control (Pastrik, 2000) in the forward Primer 2.1 primer PSA-1 5 '-gtg ggg tgg ctc CTT ga aa-3 ' Downward the primer PSA-R 5 '-tac TGA gat GTT TCA CTT ccc c-3 ' forward primer is PN-7-F 5 '-gag GCA ATA ACA ctg TGA tgc to gg-the Downward Primer 3 ' PNs-8-R 5 '-tcc GCA TCA CCT acg to gg ga-3 ' the expected Amplicon size from c. m. subsp. sepedonicus DNA matrix-502 bp (PSA-Primer pair). Expected Amplicon size from the 18s rRNA internal PCR control – 377 bp (NS-Primer pair). 2.2. PCR reaction mix reagent reagent amount final concentration of sterile upw 15.725 mkl 10 x PCR buffer 0) (15 mM MgCl2) mkl 11 x 2.5 (1.5 mM MgCl2) BSA (fraction V) (10%)
0.25% 0.1 mkl dNTP mix (20 mM) mkl 0.1 mM 0.125 Primer PSA-1 (10 mkM) 0.5 0.2 mkl mkM Primer PSA-R (10 mkM) 0.5 0.2 mkl mkM Primer NS-7-F (10 mkM) (2) 0.1 mkl mkM 0.04 Primer NS-8-R (10 mkM) (2) 0.1 mkl mkM 0.04 Taq polymerase (5 U/mkl) (1) 0.2 1.0 mkl U 5.0 mkl sample volume the total volume : 25.0 mkl (1) methods were validated using Taq polymerase, from Perkin Elmer (AmpliTaq or Gold) and Gibco BRL. (2) NS-7 F and NS-8-R ologonukleotīd concentration was optimised for potato heel end core extraction of pieces, using the homogenisation method and DNA purification according Pastrik (2000) methods. The concentrations of the reagents repeated optimization is required when used with search method or other DNA isolation method. 2.3. PCR reaction conditions make such a program: 1 cycle of 3 minutes at 950 ° C (denaturation of template DNA) 10 cycle 1 minute 950 ° C (denaturation of template DNA) 1 minute 640 ° C (for primer hybridization) 1 minute 720 ° C (extension of copy) 25 cycles 950 ° C for 30 seconds (DNA matrix denaturing) 620 ° C in 30 seconds (the primer melting) 1 minute 720 ° C (extension of copy) 1 cycle of 5 minutes 720 ° C (fragment after filling) to store 40 ° C Note : This application has been optimized for use with an MJ Research PTC 200 cycle thermostat. 2.4. restriction enzyme analysis of Amplicon From c. m. subsp. sepedonicus DNA amplified PCR products produce a distinctive restriction fragment length polimor-fism with enzyme Bgl II after 30 min incubation at 370 c. From c. m. subsp. sepedonicus acquired the characteristic restriction fragment size is 282 bp and 220 bp. 3. preparation of the sample loading buffer: 3.1. Bromphenol blue (10%-stock solution) bromphenol blue 5 g distilled water (bidest) 50 ml 3.2. sample loading buffer glycerol (86%) 3.5 ml bromphenol blue (5.1) (bidest) mkl 6.2 300 ml 4.10 x TRIS acetate EDTA (Tae) buffer, pH 8.0 TRIS buferšķīdmkM of 48.4 g 11.42 ml glacial acetic acid EDTA (disodium salt) 3.72 g distilled water 1.00 l before use, dilute 1 x. Also available in trade (e.g. Invitrogen or equivalent).
Minister of agriculture m. Roze Ministry of Agriculture presented version of annex 6 of the Cabinet of Ministers of 29 May 2007 regulations no 365-Validated reagents for FISH test oligo 1:1.1. Cms-specific probe CMS-CY3-01: ttg cgg ggc GCA 5′-ctc tgc acg-3 the cat '; The specific probe will not EUR-338-FITC: 5′-GCA to gcc tcc CGT agg AGT-3′; 2. Fixative solution: 2.1 the fixative contains paraformaldehyde, šķidum which is toxic; 2.2. to approximately 600 C heat 9 ml ultra pure water (such as grade water (UPW)) and add 0.4 g paraformaldehyde. Paraformaldehyde is dissolved, add 5 drops of 1N NaOH and stirring with a magnetic stirrer; 2.3. adjust pH to 7.0 by adding 0.1 M phosphate buffer (PB; pH 7.0) and 5 drops of 1N HCl. Check pH with indicator strips, if necessary adjust by using the HCl or NaOH; 2.3. to filter the solution through a membrane filter of 0.22 universe and protect from dust, keep the temperature up to 40 C for future use. Alternative fixative solution: 96% ethanol; 3. hybmix (3 x): 2.7 M NaCl 60 mM TRIS-HCl (pH 7.4) EDTA (sterilized by filtration and prefabricated) 15 mM if necessary, dilute 1 x 4. Hybridisation solution: 1 x hybmix solution sodium dodecyl sulphate (SDS) 0.01% probe EUB 338 5 ng/mkl probe CMSCY301 5 ng/mkl prepare the hybridisation solution according to table 1. For each slide (containing 2 different samples and duplicates) required 90 mkl hybridisation solution. table 1 suggested quantities for preparation of hybridisation mix 2 slide 8 slide sterile upw 50.1 200.4 3 x hybmix 30.0 120.0 1% SDS 0.9 3.6 probe EUB 338 (100 ng/ul) 4.5 18.0 probe CMSCY301 (100 ng/ul) 4.5 18.0 total volume (ul) 90.0 360.0 solutions containing light sensitive oligo-must be stored in the dark at-200 C. Keep away from direct sunlight or electric lighting at the time of their use. 5.0.1 M phosphate buffer, pH 7.0 g KH2PO4 Na2HPO4 8.52 5.44 g distilled water 1.00 l dissolve ingredients, check pH and autoclave 15 minutes at 121 ºc temperature.
Minister of agriculture m. Roze Ministry of Agriculture presented in annex 7 versions of the Cabinet of Ministers of 29 May 2007 regulations no 365 Eggplant culture ' to 1 Eggplant (Solanum melongen) in pasteurized seed compost solely. Transplant seedlings with fully expanded cotyledons (10 to 14 days) in pasteurized compost; 2. Eggplant plants grown in a greenhouse under the following environmental conditions: day length 14 hours or natural day length if greater temperature day 21 to 240 C night 150 C Suitable varieties of eggplants: Black Beauty Long Tom Rim vote farming Minister m. Rose proposed the Ministry of agriculture in annex 8 versions of the Cabinet of Ministers of 29 May 2007 No. 365 of the terms positive and negative controls in preparation for the most important screening tests or IF PCR or FISH 1. prepare a virulent strain of the organism in the 72-hour passage [NCPPB 4053 or PD 406] to the MTNA medium and suspend in 10 mM fosfātšķīdum for cell concentration of approx. 1 to 2 x 108 colony-forming units per ml. It is usually easy to turbid suspension with optical density of 0.20 at 600 nm. 2. Remove the heel end cores or 200 tubers of varieties with white bark, which know that they are free from the organism.
3. Process the heel ends as usual and resuspend the pellet in 10 ml. 4. prepare 10 sterile 1.5 ml mikromēģen with ul 900 of resuspended Pellet.
5. Transfer 100 ul of the organism suspension to the first microvial. Homogenizing using vortex. Prepare decimal dilutions next five microvials. The six contaminated microvials will be used as positive controls. The four contaminated microvials will be used as negative controls. Accordingly, the highlight of the Mikromēģen.
6. prepare 100 mkl testējan 1.5 ml of sample parts microvials thus obtaining nine replicas of each control. To use keep-16 to-240 c.
7. the presence of the Organism and of quantification should be first confirmed by IF.
8. PCR test perform DNA extraction from positive and negative control samples with each series of test samples.
9. IF and FISH tests to make positive and negative control test with each series of test samples.
10. IF, FISH and PCR tests for the determination of the sensitivity of the organism to the minimum of the positive controls should be 106 and 104 cells/ml, and they are not found in any of the negative controls. Minister of agriculture m. rose
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