Tilletia indica - The Food and Environment Research Agency - Defra

More documents

Recommendations

Info

inside surface of a Petri dish lid. Place the lids over Petri dish bases containing approximately 5 ml potato dextrose broth so that sporidia can then be released onto the broth surface. Incubate at 21°C with 12 hour light cycle. 12. After 2–3 days, basidiospores deposited onto the broth surface produce small mats of mycelia approximately 0.5–1.0 cm diameter. With a sterile dissecting needle, remove each mycelial mat, touching them onto sterile filter paper to remove excessive broth. Place the mycelia in suitable vials (e.g. 1.5– 2.0 ml microcentrifuge tubes) for immediate DNA extraction, or store at -80°C for subsequent DNA extraction. 1 Instead of bleach, teliospores can be surface sterilised for 30 minutes in 5–10 ml of acidic electrolysed water (AEW). AEW effectively surface sterilises teliospores but, compared to a 1–2 minute bleach treatment, AEW stimulates rather than reduces teliospore germination (Bonde et al., 1999). AEW properties: pH 2.3–2.8; redox potential (ORP) c. 1100 mv; free chlorine 10–15 ppm. 2 AWA prepared using Agar Technical No. 3 (2%) from Oxoid; antibiotics penicillin and streptomycin (60 mg penicillin-G (Na salt) & 200 mg streptomycin sulfate per litre of agar). After autoclaving, add the antibiotic suspension to the cooled agar prior to pouring plates. Figure 3.9. Tilletia indica teliospores germinating on water agar after 10–14 days, producing a tuft of primary sporidia (basidiospores) at the apex of the promycelium. Primary sporidia germinate in situ to produce small colonies which produce secondary sporidia of two types: further filiform sporidia; allantoid sporidia which are forcibly discharged. Agar plug with germinated teliospore(s) PD broth Figure 3.10. Agar plug with germinated teliospores placed on the inside of a Petri dish lid suspended over potato dextrose broth 31
Materials Aspirator bottle with distilled water 20 µm mesh nylon sieve (4 cm diameter) Sterile disposable Pasteur pipettes Pipettor (1000 µl capacity) and sterile, disposable pipettor tips (plugged) Pipettor (200 µl capacity) and sterile, disposable pipettor tips (plugged) Sterile, disposable 3 ml Pasteur pipettes 15 ml disposable conical bottom centrifuge tubes Centrifuge (to take 15 ml centrifuge tubes) Autoclavable disposable waste bottle Autoclave bags 10% Bleach solution 1 (1 part domestic bleach: 9 parts water; c. 0.3–0.5% active NaOCl) Sterile distilled water Sterile disposable spreader Antibiotic water agar (AWA) plates (2% Technical agar No.3, Oxoid; 60 mg penicillin-G (Na salt) and 200 mg streptomycin sulfate per litre of agar). Parafilm‘M’ or electrical tape Scalpel Potato dextrose broth (Difco) Dissecting needle Sterile filter paper 1.5–2.0 ml microcentrifuge tubes 1 Acidic electrolysed water (AEW) with the following properties can be used instead of 10% bleach (Bonde et al., 1999): pH 2.3–2.8; redox potential (ORP) c. 1100 mv; free chlorine (5)-10-15 ppm). Equipment: Super Oxseed Labo, Advanced H2O Inc., Alameda, CA, USA. 32
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 and 32: Sample d (✔ boxes) T. horrida T.
Page 33: C. Protocol for isolation and germi
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?