Tilletia indica - The Food and Environment Research Agency - Defra

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TILLETIA INDICA PROTOCOL DETECTION AND IDENTIFICATION OF TILLETIA INDICA (KARNAL BUNT) CONTENTS CONTENTS 2 LIST OF TABLES 3 LIST OF FIGURES 4 SECTION I: INTRODUCTION 5 Principle hosts 5 Symptoms 5 SECTION II: DIAGNOSTIC SCHEME 6 Scope of the diagnostic scheme 6 Flow diagram for diagnosis 7 Sampling 8 Detection 8 Isolation 9 Identification 9 Comparison with other similar organisms 9 Confirmation 17 Requirements for a positive diagnosis 17 Report on the diagnosis 18 Contact point for protocol 18 List of participants in the ring test 19 SECTION III: PROTOCOLS A. Protocol for extracting teliospores from seed or grain by size-selective sieving 20 B. Protocol for morphological identification 25 C. Protocol for isolation & germination of teliospores for molecular confirmation 30 D. Protocol for confirmation by traditional PCR using species specific primers ** E. Protocol for confirmation by PCR using TaqMan® ** F. Protocol for confirmation using restriction enzyme analysis ** 2
SECTION IV: REFERENCES ** 3
Page 1: EU RECOMMENDED PROTOCOL FOR THE DIA
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 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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