Tilletia indica - The Food and Environment Research Agency - Defra
Tilletia indica - The Food and Environment Research Agency - Defra
Tilletia indica - The Food and Environment Research Agency - Defra
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median profiles can be enhanced by bleaching the teliospores in 10% domestic bleach<br />
(sodium hypochlorite) for 15–20 minutes; if necessary, spores can then be rinsed twice in<br />
water <strong>and</strong> stained (e.g. trypan blue or cotton blue in lactoglycerol), (Figure 2.5). In general, T.<br />
<strong>indica</strong> teliospores have a smoother, more complete median outline due to their spines being<br />
more densely arranged; profiles of T. walkeri are more irregular with gaps between the spines<br />
due their spines being more coarsely arranged (Figure 2.5).<br />
In culture, T. walkeri <strong>and</strong> T. <strong>indica</strong> produce very similar colonies. On potato dextrose agar<br />
(PDA) after 14 days at 19°C with a 12 hour light cycle, both species typically produce white<br />
to cream-coloured, slow growing, irregular, crustose colonies, about 4–6 mm in diameter<br />
(Figure 2.6). In comparison, comparable cultures of T. horrida grow significantly more<br />
slowly (colonies only 2–3 mm in diameter) due to their higher temperature optima. T. horrida<br />
isolates may also produce a reddish-purple pigment (Figure 2.6), both on PDA <strong>and</strong> potato<br />
dextrose broth.<br />
Other tuberculate-spored <strong>Tilletia</strong> species have teliospores that can appear morphologically<br />
similar to those of T. <strong>indica</strong> (Pimentel, et al., 1998; Durán & Fischer, 1961). <strong>The</strong>se species<br />
are less likely to be found as contaminants of wheat, but they include: T. barclayana (smut of<br />
various gramineae, e.g. Panicum <strong>and</strong> Paspalum), T. eragrostidis (on Eragrostis), T. inolens<br />
(on Deyeuxia forsteri), T. rugispora (on Paspalum), T. boutelouae (on Bouteloua gracilis).<br />
None of these morphologically similar species, or T. walkeri or T. horrida, has been found to<br />
naturally infect wheat.<br />
Molecular comparisons<br />
Diagnostically significant differences exist between T. <strong>indica</strong>, T. walkeri <strong>and</strong> T. horrida in<br />
their nuclear <strong>and</strong> mitochondrial DNA. Interspecific polymorphisms have been identified<br />
using various polymerase chain reaction (PCR) methods, including RAPDs, RFLPs <strong>and</strong><br />
AFLPs (Pimentel et al., 1998; Laroche et al., 1998). In the nuclear ribosomal (rDNA) ITS1<br />
<strong>and</strong> ITS2 regions, there is a >98% similarity between T. walkeri <strong>and</strong> T. <strong>indica</strong> sequences<br />
(Levy et al., 1998). However, within the ITS1 region, T. walkeri has a diagnostically<br />
important restriction enzyme site (ScaI) that is not present with T. <strong>indica</strong>, T. horrida or other<br />
closely related species (Pimentel et al., 1998; Levy et al., 1998). With mtDNA, sequence<br />
differences have enabled species specific primers to be designed to T. <strong>indica</strong> <strong>and</strong> T. walkeri<br />
(Frederick et al., 2000). <strong>The</strong>se primers can be used in conventional PCR assays or in a<br />
TaqMan system in conjunction with a probe (Frederick et al., 2000). <strong>The</strong>re are currently no<br />
species specific primers for T. horrida, but RFLPs can be used to identify cultures (Pimental<br />
et al., 1998). If species specific primers for T. walkeri <strong>and</strong> T. <strong>indica</strong> do not give positive<br />
results on test cultures, RFLPs, RAPDs or AFLPs may be useful tools in identification<br />
(Pimental et al., 1998). See Section III, Protocols C–F for molecular tests.<br />
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