Tilletia indica - The Food and Environment Research Agency - Defra

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14. Rinse the first 500 ml flask twice with 20 ml of water and pour through the 20 µm sieve. 15. Tilt the 20µm sieve to an angle of 30−45° (Figure 3.4). Gently wash the deposit on the membrane and on the sieve walls on to one side of the membrane using distilled water or 0.01% Tween 20 detergent from an aspirator bottle or a disposable Pasteur pipette. 16. Recover the suspension that collects at the edge of the 20 µm sieve using a clean, disposable Pasteur pipette (Figure 3.5) and place the suspension in a new 15 ml disposable conical centrifuge tube. 17. Repeat ‘Steps 15–16’ until the 20 µm sieve appears clean (this may require 5–10 repeats, and typically results in a final collected volume of about 3–5 ml in the centrifuge tube). If necessary, the 20 µm sieve can be examined under a low power microscope to check for any residual teliospores. 18. Centrifuge 2 the collected suspension at 1000 x g for 3 mins. NB. Conical-bottomed tubes are recommended, as are centrifuges with swing-out arms rather than fixed arms, as these give better pellets. If debris is seen to adhere to the inside walls of the centrifuge tubes, re-suspend in 0.01% Tween 20 and repeat the centrifugation). 19. Carefully remove the supernatant using a 1 ml pipettor with a plugged, disposable pipette tip, or a new disposal Pasteur pipette. Take care not to disturb the pellet (discard the removed supernatant into a disposable waste vessel for quarantine disposal). 20. Re-suspend the pellet using distilled water to give a final volume of 50−100 µl, or more if the pellet volume requires. NB. If warm laboratory conditions cause water preparations to dry out quickly, then Shear’s solution, or just a glycerol solution, can be used as an alternative to water. However, teliospores start be killed after a few minutes exposure in Shear’s and little germination can be expected after 1 hour’s exposure; slides should be assessed immediately (within 10–20 mins) and any spores recovered immediately from the slide (see Protocol C) and washed in water to allow germination and the recommended molecular confirmations. 21. Pipette a 20 µl aliquot of the suspension onto a microscope slide and place a cover slip (18 x 18 mm) on top. 22. Examine the whole slide immediately (the slide can quickly dry out) for teliospores of T. indica (Figure 3.6) using a compound microscope at x100–x400 magnification. Assess the characteristics of any teliospores at x100 magnification. Teliospores of T. indica are mainly 25–45 µm in diameter, pale orange but mostly reddish-brown to opaque-black, and densely echinulate. (For reference, see: Section II of the Diagnostic Protocol, Table 2.2. & Figure 2.2; Section III, Protocol B). 23. Repeat ‘Step 22’ with further 20 µl aliquots until the whole suspension is examined. 24. If suspect teliospores are found, refer to, and follow, the morphological diagnostic protocol (Section III, Protocol B) and the general Diagnostic Scheme (Section II), i.e: record morphological characters (e.g. size, colour, ornamentation); examine the sample for bunted seeds; isolate & germinate teliospores for molecular confirmation, if required. 2 Equation for calculating RCF (x g) from RPM: RCF = 1.12 rmax (RPM/1000) 2 , where rmax is the radius (in mm) from the center of rotation to the bottom of the centrifuge tube. 22
25. Finally, bleach all equipment used and rinse with water before re-using (see Steps 1 & 2). Figure 3.1. Size-selective sieving wash test: 20 µm mesh nylon sieve (mounted between 4 cm diameter cylinders: left), 53 µm sieve (mounted between 11 cm diameter cylinders; right) and a 50 g grain sample in a 250 ml Erlenmeyer flask with 100 ml of 0.01% Tween-20 aqueous solution. Figure 3.2. Size-selective sieving wash test: arrangement of sieves (20 µm, sieve, left; 53 µm sieve, right) mounted in funnels over 500 ml Erlenmeyer flasks in preparation for sizeselective sieving of wash water from a 50 g grain sample. 23
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 and 20: conform to one specific species (si
Page 21: SECTION III. PROTOCOLS A1. Protocol
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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