Tilletia indica - The Food and Environment Research Agency - Defra

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Isolation Tilletia indica is a facultative biotroph. To produce cultures, teliospores are soaked in water, quickly surface sterilised and then germinated on water agar plates (Section III, Protocol C). After 7-14 days, non-dormant teliospores produce a promycelium bearing 32–128 or more basidiospores (primary sporidia) at its tip. These basidiospores can then be cultured directly on solid or liquid nutrient media. Identification The morphology of Tilletia indica is as follows (see also: Table 2.2; Figure 2.2; and Section III, Protocol B): Teliospore shape and size * : globose to subglobose, sometimes with a small hyphal fragment (more common on immature teliospores, but occasionally on mature teliospores), mostly 22– 47 µm in diameter, occasionally larger (mean 35–41 * µm). Teliospore colour: pale orange to brown to dark, reddish brown; some teliospores black and opaque (Figure 2.2). Teliospore ornamentation * : densely ornamented with sharply pointed to truncate spines, occasionally with curved tips, 1.5–5.0 µm high, which in surface view appear as either individual spines (densely echinulate) or as closely spaced, narrow ridges (finely cerebriform) (Figure 2.2).; the spines are covered by a thin hyaline membrane. Sterile cells: globose, subglobose to lacrymiform (tear-shaped), yellowish brown, 10–28 x 48 µm, with or without an apiculus (short stalk), with smooth walls up to 7 µm thick and laminated. Sterile cells are likely to be uncommon in sieved washings. * The mounting medium and heating or warming treatments can affect teliospore size (Aggarwal et al., 1990; Khanna & Payak, 1968; Castlebury & Carris, 1999). This protocol assumes that spores are mounted in water and not warmed or heated; suspect spores can then be germinated for any subsequent PCR confirmation. However, surface ornamentation can sometimes not be seen clearly in water. In such cases, mounting teliospores in lactoglycerol or Shears’s solution (Mathur & Cunfer, 1993) and gently heating the slides may improve clarity. Comparison with Other Similar Organisms Morphological comparisons Other tuberculate-spored Tilletia species may be confused with T. indica (Durán & Fischer, 1961; Durán, 1987). In particularly, the morphologically and genetically similar fungus Tilletia walkeri (ryegrass bunt), and also T. horrida (rice smut), are known contaminants of wheat seed or grain (Cunfer & Castlebury, 1999; Castlebury & Carris, 1999; Smith et al., 1996). The most important morphological characters that discriminate T. indica, T. walkeri and T. horrida are teliospore size (range and mean), exospore ornamentation and colour (Table 2.2; Figures 2.2–2.4; Section III, Protocol B). If sufficient numbers of teliospores are present, T. horrida teliospores are principally distinguished from T. indica by their smaller size, chestnut-brown colour and spines that are frequently curved and that appear as polygonal scales in surface view. T. walkeri and T. indica have a larger degree of overlap in morphological characters. However, T. walkeri teliospores are on average smaller, paler in colour (never black/opaque) and have coarser exospore ornamentation which in surface view gives the appearance of wide, incompletely cerebriform ridges or thick clumps (Castlebury & Carris, 1999). In median view, the exospore spine profiles may also aid identification. The 10
median profiles can be enhanced by bleaching the teliospores in 10% domestic bleach (sodium hypochlorite) for 15–20 minutes; if necessary, spores can then be rinsed twice in water and stained (e.g. trypan blue or cotton blue in lactoglycerol), (Figure 2.5). In general, T. indica teliospores have a smoother, more complete median outline due to their spines being more densely arranged; profiles of T. walkeri are more irregular with gaps between the spines due their spines being more coarsely arranged (Figure 2.5). In culture, T. walkeri and T. indica produce very similar colonies. On potato dextrose agar (PDA) after 14 days at 19°C with a 12 hour light cycle, both species typically produce white to cream-coloured, slow growing, irregular, crustose colonies, about 4–6 mm in diameter (Figure 2.6). In comparison, comparable cultures of T. horrida grow significantly more slowly (colonies only 2–3 mm in diameter) due to their higher temperature optima. T. horrida isolates may also produce a reddish-purple pigment (Figure 2.6), both on PDA and potato dextrose broth. Other tuberculate-spored Tilletia species have teliospores that can appear morphologically similar to those of T. indica (Pimentel, et al., 1998; Durán & Fischer, 1961). These species are less likely to be found as contaminants of wheat, but they include: T. barclayana (smut of various gramineae, e.g. Panicum and Paspalum), T. eragrostidis (on Eragrostis), T. inolens (on Deyeuxia forsteri), T. rugispora (on Paspalum), T. boutelouae (on Bouteloua gracilis). None of these morphologically similar species, or T. walkeri or T. horrida, has been found to naturally infect wheat. Molecular comparisons Diagnostically significant differences exist between T. indica, T. walkeri and T. horrida in their nuclear and mitochondrial DNA. Interspecific polymorphisms have been identified using various polymerase chain reaction (PCR) methods, including RAPDs, RFLPs and AFLPs (Pimentel et al., 1998; Laroche et al., 1998). In the nuclear ribosomal (rDNA) ITS1 and ITS2 regions, there is a >98% similarity between T. walkeri and T. indica sequences (Levy et al., 1998). However, within the ITS1 region, T. walkeri has a diagnostically important restriction enzyme site (ScaI) that is not present with T. indica, T. horrida or other closely related species (Pimentel et al., 1998; Levy et al., 1998). With mtDNA, sequence differences have enabled species specific primers to be designed to T. indica and T. walkeri (Frederick et al., 2000). These primers can be used in conventional PCR assays or in a TaqMan system in conjunction with a probe (Frederick et al., 2000). There are currently no species specific primers for T. horrida, but RFLPs can be used to identify cultures (Pimental et al., 1998). If species specific primers for T. walkeri and T. indica do not give positive results on test cultures, RFLPs, RAPDs or AFLPs may be useful tools in identification (Pimental et al., 1998). See Section III, Protocols C–F for molecular tests. 11
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: Sampling Seed lots should be sample
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 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

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