Tilletia indica - The Food and Environment Research Agency - Defra

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B. Protocol for morphological identification 1. If tuberculate teliospores are found in a wash test, record the morphological characteristics of the teliospores using Table 3.1 (refer also to Figures 2.2–2.4, 3.6 and Table 2.2). NB. Tuberculate teliospores detected in wash tests of wheat grain are assumed to be either Tilletia indica, T. walkeri or T. horrida. Other tuberculate-spored Tilletia species that infect various grasses cannot be excluded as contaminants, but have not previously been found contaminating wheat (see Section II). 2. Follow the Decision Scheme (Figure 3.8) and examine the submitted grain sample (including the 50 g washed sub-samples) for bunted wheat seeds or bunted seeds of other Poaceae, e.g. ryegrass seed (see Section II, Figure 2.1). 3. If wheat seeds with Karnal bunt symptoms are found, confirm Tilletia indica by microscopic examination of the teliospores in the seed (Section II; Table 2.2; Table 3.2). 4. If bunted ryegrass seeds are found, but no bunted wheat seeds, confirm T. walkeri by microscopic examination of the teliospores in the seed (Table 3.2). If confirmed, compare teliospores from the seed with those found in the wash test. If the teliospores are identical make a diagnosis. Molecular confirmation of the teliospores from the wash test is still recommended. 5. If wheat seeds infected with T. indica or ryegrass seeds infected with T. walkeri are not found, make a presumptive identification of teliospores found in the wash test: use Table 3.2, in conjunction with the following ‘guiding diagnostic principles’ (adapted from NAPPO, 1999): • Samples with teliospores all less than 36 µm, with curved spines, are most likely to be T. horrida. • Samples with teliospores >36 µm are most likely to be T. indica. • Samples with teliospores mostly (28–35 µm), translucent brown, never black/opaque, very spherical, with blunt spines with distinct gaps between (made more obvious in profile after bleaching) are most likely to be T. walkeri, especially if grain is from areas where ryegrass is grown alongside wheat or where ryegrass seeds are present in the sample. • Samples with mature, dark teliospores less than 25 µm are most likely to be T. horrida not T. walkeri or T. indica. • Samples with some black, opaque teliospores are most probably T. indica (T. walkeri teliospores are never opaque, black; T. horrida teliospores can be dark, semi-opaque). 6. If relatively large numbers of teliospores are present (e.g. ≥10 spores), it may be possible to identify the teliospores morphologically if all morphological criteria (size range; mean size; colour; ornamentation) clearly conform to any one species (see Table 3.2). However, molecular confirmation is still recommended if bunted wheat seeds were not found in the grain sample. 7. If only a few teliospores are detected (e.g.
Teliospore Size (µm) Colour Ornamentation Notes Number (diam.) (see codes below) (see codes below) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 etc Size range Mean ± s.d Provisional identification Colour code examples Ornamentation code examples BO Black/opaque DE Densely echinulate (spines densely and individually arranged) RB Reddish-brown FC Finely cerebriforn (spines forming closely spaced narrow ridges) CB Chestnut-brown CC Coralloid (ridges much branched) P (PY, PO, PB) Pale (yellow/orange/brown) CO Coarsely cerebriform (spines coarsely arranged forming wide, incompletely cerebriform ridges) TC Thick clumps (spines forming thick clumps) PS Polygonal scales (curved in profile) Table 3.1. Example of a record sheet that could be used for teliospores detected in wash tests. 29
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: A2. Protocol for extracting teliosp
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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