Tilletia indica - The Food and Environment Research Agency - Defra

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
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