Tilletia indica - The Food and Environment Research Agency - Defra

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LIST OF PARTICIPANTS IN THE RING TEST (confirmed participation, as of 6 th December 2001) Anna Radova State Phytosanitary Administration Division of Diagnostics Slechtitelu 11 78371 Olomouc CZECH REPUBLIC Irene Vloutoglou Benaki Phytopathological Institute Plant Pathology Department 8, S. Delta Street 14561 Kifissia, Athens GREECE Angelo Porta-Puglia Istituto Sperimentale per la Patologia Vegetale, Via C.G. Bertero, 20 I-00156 Rome ITALY Carla Montuschi Servizio Fitosanitario Regionale Via Corticella, 133 40129, Bologna, ITALY Ilse van Browershaven Plantenziektenkundige Dienst Postbus G102 6700 HC Wageningen NETHERLANDS Maria de Jesus Gomes; E. Diogo and Mª R. Malheiros Direcção-Geral de Protecção das Culturas (DGPC) Edificio 1 - Tapada da Ajuda 1349-018 Lisboa PORTUGAL Valerie Cockerell Scottish Agricultural Science Agency East Craigs Edinburgh EH12 8NJ SCOTLAND Ann Barnes Central Science Laboratory, Sand Hutton, York YO41 1LZ UNITED KINGDOM 20
SECTION III. PROTOCOLS A1. Protocol for extracting teliospores from untreated seed or grain by size-selective sieving (based on Peterson et al., 2000) Method 1. Bleach the sieves, funnels and flasks by immersion for 15 minutes in 30% bleach. 1 2. Rinse the bleach thoroughly from the equipment with tap water. 3. Weigh 50 g of grain into a new, disposable, large weigh boat. (See Section II, Table 2.1, for the number of 50 g sub-samples required to detect different levels of contamination; 3 replicates detects a level of 1 spore per 50 g sample with a 99% confidence). 4. Pour the 50 g sub-sample of grains into a 250 ml Erlenmeyer flask (Figure 3.1) 5. Add 100 ml of 0.01% Tween-20 aqueous solution to the flask. Seal the top of the flask (e.g. with Parafilm or clingfilm). 6. Place the flask on a flask shaker set at an appropriate speed (e.g. 350 oscillations/minute, or 200 r.p.m for an orbital shaker) to ensure good agitation for 3 minutes to release any teliospores from the grain. Alternatively the flask can be shaken or swirled by hand. 7. Place a 53 µm mesh nylon sieve (11 cm diameter) in a funnel over a clean 500 ml Erlenmeyer flask (Figure 3.2) then pour the whole contents of the flask (the grain and the wash water) into the sieve (Figure 3.3). 8. Rinse the 250 ml flask with 20–50 ml of distilled water from an aspirator bottle, then pour evenly over the grain on the 53 µm sieve. 9. Repeat ‘Step 8’ twice more. 10. Thoroughly rinse the grain on the 53 µm sieve by washing with further distilled water from an aspirator bottle (Figure 3.3) to give a final collected volume of c. 300–400 ml. 11. Remove the 53 µm sieve from the funnel and rinse the funnel with two aliquots of 10–20 ml of distilled water, collecting the water in the same 500 ml flask. (NB. Keep the washed grain sample(s) and also the remainder of the submitted sample that has not been tested, in case there is a need to examine grain directly for disease symptoms – see Protocol B). 12. Place a 20 µm mesh nylon sieve (4 cm diameter; Figure 3.2) in a funnel over a second 500 ml Erlenmeyer flask. 13. Pour the collected washings from Steps 7–11 through the 20 µm nylon sieve. NB. Wet the sieve membrane prior to use and gently tap the outside of the PVC sieve-holder repeatedly to facilitate a good rate of sieving; otherwise the membrane can quickly become blocked. 1 Bleach eliminates the risk of false positives by cross contamination from previous samples; bleach kills teliospores and makes them appear hyaline compared with the normally dark, pigmented spores. The bleach solution should be changed regularly, as appropriate. 21
Page 1 and 2: EU RECOMMENDED PROTOCOL FOR THE DIA
Page 3 and 4: SECTION IV: REFERENCES ** 3
Page 5 and 6: LIST OF FIGURES Figure 1.1. Grain i
Page 7 and 8: SECTION II: DIAGNOSTIC SCHEME Scope
Page 9 and 10: Sampling Seed lots should be sample
Page 11 and 12: median profiles can be enhanced by
Page 13 and 14: Figure 2.2. Teliospores of Tilletia
Page 15 and 16: Figure 2.4. Teliospores of Tilletia
Page 17 and 18: 7 days 10 days 14 days T. horrida T
Page 19: conform to one specific species (si
Page 23 and 24: 25. Finally, bleach all equipment u
Page 25 and 26: Figure 3.6. Tilletia indica teliosp
Page 27 and 28: A2. Protocol for extracting teliosp
Page 29 and 30: Teliospore Size (µm) Colour Orname
Page 31 and 32: Sample d (✔ boxes) T. horrida T.
Page 33 and 34: C. Protocol for isolation and germi
Page 35 and 36: Materials Aspirator bottle with dis
Page 37 and 38: SECTION IV: REFERENCES Aggarwal, R,

Tilletia indica - The Food and Environment Research Agency - Defra

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