Tilletia indica - The Food and Environment Research Agency - Defra

More documents

Recommendations

Info

Materials 30% Bleach solution (3 parts household bleach: 7 parts water; c. 1.6% active NaOCl) Wash water = 0.01% aqueous Tween-20 (detergent) Large weigh boats (8 x 8 cm) Weighing balance 250 ml Erlenmeyer glass flask 100 ml measuring cylinder Parafilm‘M’ or clingfilm Laboratory flask shaker (alternatively, shaking flasks by hand is acceptable) 500 ml Erlenmeyer glass flask x 2 Funnel (approx. 13 cm diameter) 53 µm mesh nylon sieve (11 cm external diameter): mesh from Spectrum Laboratories * 20 µm mesh nylon sieve (4 cm external diameter): mesh from BDH or Spectrum Laboratories Aspirator bottle with distilled water Sterile disposable 3 ml Pasteur pipettes Pipettor (100 µl capacity) plus disposable pipettor tips (plugged) Pipettor (1000 µl capacity) plus disposable pipettor tips (plugged) 15 ml sterile, disposable conical bottom centrifuge tubes Centrifuge (to take 15 ml centrifuge tubes above) Autoclavable, disposable waste bottle Autoclave bags Glass microscope slides (76 x 21 mm) Microscope cover slips (18 x 18 mm) Compound microscope (x 100–400 magnification) Dissecting microscope (x 10–70 magnification) Shear’s solution † (as an alternative mounting medium to water if slides are prone to drying; however, Shear’s starts to kill teliospores after a few minutes exposure and little germination can be expected after 1 hours exposure) * If 53 µm nylon mesh cannot be sourced, then an alternative mesh size could be used, e.g. 50 or 70 µm mesh † Shear’s solution: 300 ml Mc Ilvaine’s buffer †† 6 ml Potassium acetate 120 ml Glycerine 180 ml Ethyl alcohol (95%) †† Prepare Mc Ilvaine’s buffer (Mathur & Cunfer, 1993) as follows: Dissolve 19.212 g citric acid (C6H8O7) in 1000 ml distilled water and mix thoroughly. Dissolve 28.392 g of disodium phosphate (Na2HPO4) in 1000 ml of distilled water and mix thoroughly. Mix 8.25 ml of citric acid solution with 291.75 ml of disodium phosphate solution and mix thoroughly. 26
A2. Protocol for extracting teliospores from fungicide treated seed by size-selective sieving (adapted from Agarwal & Mathur, 1992 and based on Peterson et al., 2000) Method (added after the ring test, therefore not ring tested) 1. Follow Steps 1–4 from Protocol A1. 2. To the 50 g fungicide treated sample add 100 ml of 0.2% (or 1%) sodium hydroxide 1 (NaOH) and incubate for 24 hours. 3. Add 100 ml of 0.01% Tween-20 aqueous solution to the flask (as in Step 5 of Protocol A1). Seal the top of the flask (e.g. with Parafilm or clingfilm). 4. Continue with Steps 6–25 from Protocol A1. 1 NaOH can help to remove most of the fungicide treatment allowing subsequent sieving. Without the NaOH treatment, the 20 µm sieve may become blocked by the fungicide treatment. The NaOH treatment does not affect teliospore size and colour characteristics, but does kill the teliospores (Bowyer & Inman, unpublished). An alternative to using NaOH is to use just the 53µm sieve and not the 20µm sieve, if this becomes easily blocked. 27
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: Figure 3.6. Tilletia indica 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

Create successful ePaper yourself

Delete template?

Save as template?