Tilletia indica - The Food and Environment Research Agency - Defra

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Sample declared healthy IDENTIFICATION based on teliospore morphology (Protocol B) Wash test for tuberculate Tilletia teliospores (Size selective sieving on 50 g sub-samples) (Protocol A) negative positive Many teliospores present (c. ≥10) MORPHOLOGICAL DIAGNOSIS of teliospores (Protocol B) MOLECULAR CONFIRMATION TESTS Isolate & germinate suspect teliospores to produce cultures for molecular confirmation tests (Protocol C) MOLECULAR CONFIRMATION: Restriction enzyme analysis (Protocol D) PCR with species specific primers (Protocol E) TaqMan with species specific primers & probe (Protocol F) IDENTIFICATION OF SPECIES based on morphological and molecular analyses CONFIRMATION: microscopic examination of teliospores from bunted seeds compared to those in wash test (Protocol B) Bunted seeds found Examine sample for bunted cereal seeds or bunted seeds of other Poaceae No bunted cereal grains found Few teliospores present (c.
Sampling Seed lots should be sampled according to current ISTA rules. Grain, e.g. for feed or processing, is typically more difficult to sample because consignments are usually very large and transported or stored as large, loose bulks. However, for monitoring purposes, grain should be sampled in an appropriate fashion to produce a 1–2 kg thoroughly mixed sample representative of the consignment. Detection For quarantine purposes, detection of T. indica is best achieved by a wash test (CABI/EPPO, 1997); infected parts of the grain typically disintegrate so that the teliospores contaminate other grains in the lot. The most efficient and rapid wash test method for detecting teliospores in a sample is a size-selective sieving and centrifugation technique (Section III, Protocol A; Peterson et al., 2000). This method has, on average, an 82% efficiency of recovery and microscopic examinations typically require only a few slides per 50 g sub-sample. The number of replicate 50 g sub-samples needed to detect differing levels of contamination is given in Table 2.1. Contamination level (No. of spores / 50 g sample) No. of replicate samples required for detection according to level of confidence (%) 99 % 99.9 % 99.99 % 1 3 5 6 2 2 3 4 5 1 1 1 Table 2.1. The number of replicate 50 g sub-samples needed to detect differing levels of contamination with specified confidences, assuming an equal distribution of teliospores (Peterson et al., 2000; Inman & Bowyer, EU SMT4-CT98-2252 evaluation, 2000). Direct visual examinations for bunted kernals or teliospores contaminating seed surfaces are not considered reliable methods for quarantine purposes. However, Karnal bunt may be detected by visual examination with the naked eye and low power microscopy (x10–x70 magnification). To help visualise symptoms, seed can be soaked in 0.2% sodium hydroxide for 24 hours at 20°C; this is especially useful for chemically treated seed lots where coloured dyes may obscure symptoms (Mathur & Cunfer, 1993; Agarwal & Mathur, 1992). With severe contamination, teliospores may be seen on the surface of seeds (Mathur & Cunfer, 1993). 9
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: SECTION II: DIAGNOSTIC SCHEME Scope
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 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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