Tilletia indica - The Food and Environment Research Agency - Defra

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Bunted wheat seeds found Bunted wheat NOT found; Bunted ryegrass found CONFIRMATION Karnal bunt (T. indica) confirmed by microscopic examination of teliospores from grains (Table 2.2 & 3.2) Molecular confirmation of teliospores in wash tests recommended in case of mixed species populations ALL morphological characters satisfy one specific Tilletia species (see Table 3.2) MORPHOLOGICAL IDENTIFICATION Tuberculate teliospores detected in wash test: record morphological characters (Table 3.1) Examine grain sample* and the washed sub-samples directly for bunted seeds of wheat or other Poaceae (e.g. ryegrass) CONFIRMATION Ryegrass bunt (T. walkeri) confirmed if teliospores from bunted seeds and wash test are identical and conform totally to T. walkeri (Table 2.2 & 3.2) Many teliospores present (≥10) NOT all morphological characters satisfy one Tilletia species (see Table 3.2) PRESUMPTIVE IDENTIFICATION Bunted seeds NOT found PRESUMPTIVE IDENTIFICATION Assess morphological characters of teliospores i n wash test (Table 3.1) Few teliospores present (
C. Protocol for isolation and germination of teliospores for molecular confirmation Method 1. Recover suspect teliospores from both the microscope slide and the cover slip by washing them with distilled water over a clean 20 µm sieve. Recover the teliospores from the sieve (see Protocol A, step 15–17), pipetting the suspension into a new 15 ml conical centrifuge tube. Make up the final volume to 3–5 ml with distilled water. 2. Incubate the teliospore suspension overnight at 21°C to hydrate the teliospores and make fungal and bacterial contaminants more susceptible to subsequent surface sterilisation. 3. After incubating overnight, pellet the teliospores by centrifuging at 1200 x g for 3 mins. 4. Remove the supernatant with a pipettor with a plugged tip, or a disposable Pasteur pipette, taking care not to disturb the pellet; pipette the supernatant into a suitable disposable waste bottle for subsequent quarantine disposal. 5. Resuspend the pellet in 5 ml of 10% domestic bleach 1 (c. 0.3–0.5% active NaOCl); replace the centrifuge tube cap and immediately invert the tube three time to ensure that the bleach contacts all inner surfaces. 6. Immediately centrifuge at 1200 x g for 1 minute, then quickly and aseptically remove the supernatant and resuspend the pellet in 1 ml sterile distilled water (SDW) to wash the pellet. NB. Some teliospores can be killed if the total time in the bleach exceeds 2 minutes 1 . 7. Centrifuge at 1200 x g for 5 minutes, aseptically remove the supernatant and then add another 1 ml SDW to wash the pellet again. 8. Centrifuge at 1200 x g for 5 minutes, aseptically remove the supernatant and resuspend the final pellet in 1 ml SDW. 9. Transfer 200 µl of the teliospore suspension aseptically onto individual 2% water agar plates with antibiotics 2 (AWA) and spread with a sterile spreader. (NB. 3-day-old plates are recommended as these quickly absorb the suspension; excessive surface water can inhibit teliospore germination. Alternatively, prepare the agar plates on the day of use, but pour the liquid agar when cool and do not replace the lids fully until the agar has set.) 10. Incubate the AWA plates at 21°C with a 12 hour light cycle (e.g. TLD 18W/83 Philips white light tubes). Leave for about 5 days before sealing plates with parafilm or electrical tape, or placing the plates inside clear polyethylene bags. 11. After 7–14 days, examine the plates for germinated teliospores bearing a tuft of filiform basidiospores (primary sporidia), or small colonies forming around germinated teliospores (Figure 3.9). These colonies produce secondary sporidia typically of two types: filiform and allantoid. Cut out small blocks of agar (c. 5x5 to 10x10 mm square) bearing germinated teliospores or colonies; invert the agar blocks and stick them onto the 30
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 and 22: SECTION III. PROTOCOLS A1. Protocol
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: Sample d (✔ boxes) T. horrida T.
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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