Tilletia indica
2004; Wiley; Volume: 34; Issue: 2 Linguagem: Italiano
10.1111/j.1365-2338.2004.00723.x
ISSN1365-2338
Tópico(s)Mycorrhizal Fungi and Plant Interactions
ResumoEPPO BulletinVolume 34, Issue 2 p. 219-227 Diagnostic protocols for regulated pests†Free Access Tilletia indica First published: 10 September 2004 https://doi.org/10.1111/j.1365-2338.2004.00723.xCitations: 2 European and Mediterranean Plant Protection Organization PM 7/29(1) Organisation Européenne et Méditerranéenne pour la Protection des Plantes AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Specific scope This standard describes a diagnostic protocol for Tilletia indica. Specific approval and amendment This Standard was developed under the EU DIAGPRO Project (SMT 4-CT98-2252) by partnership of contractor laboratories and intercomparison laboratories in European countries. Approved as an EPPO Standard in 2003-09. Introduction Tilletia indica causes the disease Karnal bunt, or partial bunt, of wheat (Triticum spp.). Triticale (X Triticosecale) is also naturally infected and rye (Secale) is a potential host. T. indica was added to the EC Plant Health Directive 77/93/EEC (now 2000/29/EC) as a I/AI pest in 1996 and phytosanitary requirements applied to seed and grain of Triticum, Secale and X Triticosecale imported from countries where T. indica is known to occur. Identity Name: Tilletia indica Mitra. Synonyms: Neovossia indica (Mitra) Mundkur. Taxonomic position: Fungi: Basidiomycota: Ustilaginales. Bayer computer code: NEOVIN. Phytosanitary categorization: EPPO A1 list, no. 23; EU Annex designation I/AI. Detection Symptoms T. indica is a floret-infecting fungal smut pathogen. Unlike systemic smuts, not all the seeds on an ear are usually infected. Seeds are infected through the germinal end of the grain and the fungus develops within the pericarp where it produces a powdery, brownish-black mass of teliospores. When fresh, the spore masses produce a foetid, decaying fish-like smell (trimethylamine). Seeds are usually only partially colonized, showing various degrees of infection. Point infections are most common, but infection may also spread down the adaxial groove and, in severe cases, the whole grain may appear bunted (Web Fig. 1). 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. For phytosanitary 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 (Appendix 1; 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 subsample. The number of replicate 50 g subsamples needed to detect differing levels of contamination is given in Table 1. Table 1. Number of replicate 50 g subsamples 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) No. of replicate samples required for detection according to level of confidence (%) Contamination level (no. of spores per 50 g sample) 99% 99.9% 99.99% 1 3 5 6 2 2 3 4 5 1 1 1 Direct visual examinations for bunted kernels or teliospores contaminating seed surfaces are not considered reliable methods for phytosanitary purposes. However, Karnal bunt may be detected by visual examination with the naked eye and low power microscopy (× 10–× 70 magnification). To help visualize symptoms, seed can be soaked in 0.2% NaOH for 24 h 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). Identification Isolation T. indica is a facultative biotroph. To produce cultures, teliospores are soaked in water, quickly surface-sterilized and then germinated on water-agar plates (Appendix 4). 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. Morphology Teliospores 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); pale orange to brown to dark, reddish brown; some teliospores black and opaque (Web Fig. 3); 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) (Web Fig. 3); the spines are covered by a thin hyaline membrane. Sterile cells: globose, subglobose to lacrymiform (tear-shaped), yellowish brown, 10–28 × 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. See also CMI (1983), Table 2, Web Fig. 3, Appendix (3)22 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. . Table 2. Morphological characteristics of Tilletia indica, Tilletia walkeri and Tilletia horrida Teliospore character Tilletia indica 1 Tilletia walkeri 2 Tilletia horrida 3 Size (range) µm 22–47–(61)(26–55(−64)) (23–45) 17–36(20–38(−41)) Size (mean) µm 35–41 30–31* 24–28‡ (40–44)† (34–36)† (28)† Colour Pale orange to mainly dark, reddish brown to opaque-black Pale yellow to mainly dark reddish brown (never opaque) Pale yellow to mainly light or dark chestnut brown (semiopaque) Exospore ornamentationin median view (Hawksworth et al., 1995) Sharply pointed to truncate spines (occasionally curved), 1.5–5.0 µm high, covered with a hyaline sheath Conical to truncate spines (occasionally curved), 3–6 µm high, covered with a hyaline to yellowish-brown sheath Sharply pointed or curved spines, 1.5–4.0 µm high, becoming truncated scales with maturity, covered with a hyaline to tinted sheath‡ Exospore ornamentation in surface view Spines densely arranged, either individually (densely echinulate) or in closely spaced, narrow ridges (finely cerebriform) Spines coarsely arranged, forming wide, incompletely cerebriform (to coralloid) ridges or thick clumps Spines appearing as polygonal scales (occasionally spines forming cerebriform ridges or small clumps) 1 Authors’ data. 2 Based on: Castlebury & Carris (1999); Cunfer & Castlebury (1999); Milbrath et al. (1998); Castlebury (1998). 3 As T. barclayana: Castlebury & Carris (1999); CMI Description no. 75 (1965); Durán (1987); Durán & Fischer (1961) Or as T. horrida: Aggarwal et al. (1990); Khanna & Payak (1968); Castlebury (1998). † Castlebury & Carris (1999) report larger spore sizes (in brackets) for teliospores warmed overnight at 45 °C in Shear's solution; Castlebury (1998) also reports larger teliospore sizes. * Milbrath et al. (1998), supported by Author's data from teliospores ex. Lolium (two isolates ex. Oregon, USA) in water. ‡ Author's data from teliospores ex. Oryza (California, USA; Arkansas, USA) in water; though not reported in the literature, some spores may have ridges in addition to individual spines (see Web Fig. 5). Diagnostic scheme The diagnostic scheme for T. indica, as presented in Fig. 2, describes procedures for detection of teliospores in imported seeds or grain of wheat by a size-selective sieving wash test; morphological identification of teliospores detected in wash tests; isolation and germination of teliospores for molecular confirmation and molecular confirmation of cultures. Figure 2Open in figure viewerPowerPoint Decision scheme for the detection and identification of Tilletia indica. Confirmation Morphological confirmation If only a few teliospores are present (< 10 teliospores) in a wash test, morphological characters are not considered totally reliable for confident discrimination between T. indica and the morphologically similar species that are known contaminants of wheat grain, e.g. T. walkeri and T. horrida (Table 2; Web Figs 3–5). However, if large numbers of teliospores are present (> 10 spores) then morphological identification may be possible (Appendix 3). The most important discriminatory characters are teliospore size (maximum size and mean), exospore ornamentation and colour (Table 2; Web Figs 3–5; Appendix 3). However, molecular confirmation tests are still recommended. When suspect teliospores are found in a wash test, the grains in both the washed subsample(s) and the larger submitted sample should be examined for Karnal bunt symptoms. If symptoms are found, these should be confirmed by microscopic examination of the teliospores. Any grass seeds found in the sample should also be examined for signs of bunt infection and, if found, the associated teliospores should be examined microscopically. Molecular confirmation tests are also recommended. Molecular confirmation There are three main molecular methods available to confirm presumptive morphological diagnoses: (1) restriction enzyme analysis of the ITS1 region (rDNA Internally Transcribed Spacer Region 1) after PCR application using universal ITS primers; (2) conventional PCR (polymerase chain reaction) assay using species specific primers; (3) PCR assay using species specific primers and a fluorescent probe in a TaqMan system. All these molecular confirmation methods require that teliospores are germinated and cultures produced from the resulting sporidia (Appendix 4) Detailed descriptions of these methods are still in preparation. Although PCR has been used on ungerminated teliospores, this typically requires large numbers of teliospores and even then negative results are considered unreliable (Smith et al., 1996; McDonald et al., 1999). Possible confusion with similar species 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 Tilletia 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 233 The literature on spore sizes is often variable. Spore size is affected by the mounting medium and by heating treatments. For rice smut (T. horrida, synonym T. barclayana), data from rice is potentially more reliable than data based on T. barclayana sensu lato from various Poaceae as the latter is considered a species complex. The rice pathogen is considered distinct from those on Paspalum and Panicum, but it is not known whether it is distinct from T. barclayana s.s. on Pennisetum (Pimentel, 1998; Castlebury, 1998). ; Web Figs 3–5; Appendix 3). 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 median profiles can be enhanced by bleaching the teliospores in 10% sodium hypochlorite for 15–20 min. If necessary, spores can then be rinsed twice in water and stained, e.g. with trypan blue or cotton blue in lactoglycerol (Web Fig. 6). 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 (Web Fig. 6). 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-h light cycle, both species typically produce white to cream-coloured, slow-growing, irregular, crustose colonies, about 4–6 mm in diameter (Web Fig. 7). 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 (Web Fig. 7), 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: Tilletia barclayana (smut of various Poaceae, e.g. Panicum and Paspalum), Tilletia eragrostidis (on Eragrostis), Tilletia inolens (on Lachnagrostis filiformis), Tilletia rugispora (on Paspalum), Tilletia boutelouae (on Bouteloua gracilis). None of these morphologically similar species, or T. walkeri or T. horrida, has been found naturally to 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 (Pimentel 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). Requirements for a positive diagnosis The procedures for detection and identification described in this protocol, and the decision scheme in Fig. 2, should have been followed. If bunted grains are present, a positive diagnosis can be made if the symptoms are confirmed by the presence of teliospores that are morphologically consistent with those described for T. indica in this protocol (Table 2; Appendix 3). If bunted grains are not present, the wash test and procedures for detection described in this protocol should be followed. Since there is considerable overlap in morphological characters between T. indica and various other tuberculate-spored species which can potentially contaminate seed or grain, namely T. walkeri and T. horrida, morphological identification of teliospores is only possible when a large number of teliospores are present (> 10 spores). In such cases, teliospores may be identified morphologically, but only if all key characters conform to one specific species (size range, size mean, colour and exospore ornamentation patterns: (see Appendix 3). Molecular confirmation is still recommended. If too few spores are present (< 10 spores), it may not be possible to discriminate species using morphological characters. In such cases, molecular confirmation by PCR using species – specific primers combined as appropriate with restriction – enzyme analysis is recommended for a positive diagnosis. Report on the diagnosis A report on the diagnosis should include the following information: • results obtained by the recommended procedures • the origin (country; state) and type (host; commodity) of the infected material • quantity of consignment or lot • degree of infection/contamination (e.g. the number of positive subsamples and the estimated number of teliospores detected in each positive subsample) • a description of any disease symptoms (preferably including photographs) • a description of the teliospores (with colour photographs if possible) compared with descriptions of T. indica and morphologically similar species, e.g. T. walkeri and T. horrida • if cultures were obtained for molecular confirmation, a description of the colony morphology, especially any pigmentation, and growth rate under defined conditions Cultures should be kept (mycelium from broths or mycelial plugs from agar plates can be stored frozen at −80 °C) • if molecular confirmation tests were done, a copy of the molecular test result along with positive and negative controls should be provided. For TaqMan assays, the CT-value should be noted • comments as appropriate on the certainty or uncertainty of the identification. When cultures have been obtained, these should be cultured onto potato dextrose agar (PDA) and 5-mm diameter mycelial plugs stored at −80 °C in sterile tubes. Similarly, any DNA extractions should be stored at −80 °C. Further information Further information on this organism can be obtained from: Pest and Disease Identification Team, Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom. Footnotes 1 The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO website http://www.eppo.org. 2 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. 3 The literature on spore sizes is often variable. Spore size is affected by the mounting medium and by heating treatments. For rice smut (T. horrida, synonym T. barclayana), data from rice is potentially more reliable than data based on T. barclayana sensu lato from various Poaceae as the latter is considered a species complex. The rice pathogen is considered distinct from those on Paspalum and Panicum, but it is not known whether it is distinct from T. barclayana s.s. on Pennisetum (Pimentel, 1998; Castlebury, 1998). 4 A. Radova, State Phytosanitary Administration, Olomouc, CZ; I. Vloutoglou, Benaki Phytopathological Institute, Athens (GR); A. Porta-Puglia, Istituto Sperimentale per la Patologia Vegetale, Rome (IT); C. Montuschi, Servizio Fitosanitario Regionale, Bologna (IT); I. van Browershaven, Plantenziektenkundige Dienst, Wageningen (NL); M. de Jesus Gomes, E. Diogo & M.R. Malheiros, Direcção-Geral de Protecção das Culturas, Lisboa (PT); V. Cockerell, Scottish Agricultural Science Agency, East Craigs, Edinburgh (GB); A. Barnes, Central Science Laboratory, Sand Hutton, York (GB). 5 Shear's solution: 300 mL Mc Ilvaine's buffer, 6 mL Potassium acetate, 120 mL Glycerine 180 mL Ethyl alcohol (95%). Prepare Mc Ilvaine's buffer (Mathur & Cunfer, 1993) as follows: dissolve 19.212 g citric acid in 1000 mL distilled water and mix thoroughly; dissolve 28.392 g of disodium phosphate (Na2HPO4) in 1000 mL of distilled water and mix thoroughly; mix 8.25 mL of citric acid solution with 291.75 mL of disodium phosphate solution and mix thoroughly. 6 Bleach eliminates the risk of false positives by cross contamination from previous samples; bleach kills teliospores and makes them appear hyaline compared with the normally dark, pigmented spores. The bleach solution should be changed regularly, as appropriate. 7 Conical-bottomed tubes are recommended, as are centrifuges with swing-out arms rather than fixed arms, as these give better pellets. If debris is seen to adhere to the inside walls of the centrifuge tubes, re-suspend in 0.01% Tween 20 and repeat the centrifugation 8 If warm laboratory conditions cause water preparations to dry out quickly, then Shear's solution, or just a glycerol solution, can be used as an alternative to water. However, teliospores start be killed after a few minutes exposure in Shear's and little germination can be expected after exposure of 1 h. Slides should be assessed immediately (within 10–20 min) and any spores recovered immediately from the slide (see Appendix 4) and washed in water to allow germination and the recommended molecular confirmations 9 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 Fig. 2 10 Acidic electrolysed water (AEW) with the following properties can be used instead of 10% bleach (Bonde et al., 1999): pH 2.3–2.8; redox potential (ORP) c. 1100 mv; free chlorine (5)–10–15 (ppm). Equipment: Super Oxseed Labo, Advanced H2O Inc., Alameda, US. 11 Instead of bleach, teliospores can be surface sterilized for 30 min in 5–10 mL of AEW (see above). AEW effectively surface sterilizes teliospores but, compared to a 1–2 min bleach treatment, stimulates rather than reduces teliospore germination (Bonde et al., 1999). NB. Some teliospores can be killed if the total time in the bleach exceeds 2 min 12 3-day-old plates are recommended, as these quickly absorb the suspension; excessive surface water can inhibit teliospore germination. Alternatively, prepare the agar plates on the day of use, but pour the liquid agar when cool and do not replace the lids fully until the agar has set. Acknowledgements This protocol was originally drafted by: A. J. Inman, K. J. D. Hughes and R. J. Bowyer, Central Science Laboratory, York (GB). This protocol was ring-tested in different European laboratories44 A. Radova, State Phytosanitary Administration, Olomouc, CZ; I. Vloutoglou, Benaki Phytopathological Institute, Athens (GR); A. Porta-Puglia, Istituto Sperimentale per la Patologia Vegetale, Rome (IT); C. Montuschi, Servizio Fitosanitario Regionale, Bologna (IT); I. van Browershaven, Plantenziektenkundige Dienst, Wageningen (NL); M. de Jesus Gomes, E. Diogo & M.R. Malheiros, Direcção-Geral de Protecção das Culturas, Lisboa (PT); V. Cockerell, Scottish Agricultural Science Agency, East Craigs, Edinburgh (GB); A. Barnes, Central Science Laboratory, Sand Hutton, York (GB). . Appendices Appendix 1. Method for extracting teliospores from untreated seed or grain by size-selective sieving (based on Peterson et al., 2000) Materials 30% bleach solution (3 parts household bleach: 7 parts water; c. 1.6% active NaOCl); wash water = 0.01% aqueous Tween-20 (detergent); large weigh boats (8 × 8 cm); weighing balance; 250-mL Erlenmeyer glass flask; 100-mL measuring cylinder; Parafilm'M′ or clingfilm; laboratory flask shaker (alternatively, shaking flasks by hand is acceptable); 500-mL Erlenmeyer glass flask × 2; funnel (approx. 13 cm diameter); 53 (50 or 70) µm mesh nylon sieve (11 cm external diameter) mesh from Spectrum Laboratories; 20-µm mesh nylon sieve (4 cm external diameter): mesh from BDH or Spectrum Laboratories; aspirator bottle with distilled water; sterile disposable 3 mL Pasteur pipettes; pipettor (100-µL capacity) plus disposable pipettor tips (plugged); pipettor (1000-µL capacity) plus disposable pipettor tips (plugged); 15-mL sterile, disposable conical bottom centrifuge tubes; centrifuge (to take 15-mL centrifuge tubes above); autoclavable, disposable waste bottle; autoclave bags; glass microscope slides (76 × 21 mm); microscope cover slips (18 × 18 mm); compound microscope (× 100–400 magnification); dissecting microscope (× 10–70 magnification); Shear's solution55 Shear's solution: 300 mL Mc Ilvaine's buffer, 6 mL Potassium acetate, 120 mL Glycerine 180 mL Ethyl alcohol (95%). Prepare Mc Ilvaine's buffer (Mathur & Cunfer, 1993) as follows: dissolve 19.212 g citric acid in 1000 mL distilled water and mix thoroughly; dissolve 28.392 g of disodium phosphate (Na2HPO4) in 1000 mL of distilled water and mix thoroughly; mix 8.25 mL of citric acid solution with 291.75 mL of disodium phosphate solution and mix thoroughly. (as an alternative mounting medium to water if slides are prone to drying; however, Shear's starts to kill teliospores after a few minutes exposure and little germination can be expected after exposure of 1 h). Method Bleach the sieves, funnels and flasks by immersion for 15 min in 30% bleach66 Bleach eliminates the risk of false positives by cross contamination from previous samples; bleach kills teliospores and makes them appear hyaline compared with the normally dark, pigmented spores. The bleach solution should be changed regularly, as appropriate. . Rinse the bleach thoroughly from the equipment with tap water. Weigh 50 g of grain into a new, disposable, large weigh boat (see Fig. 2, Table 1, for the number of 50 g subsamples required to detect different levels of contamination; 3 replicates detects a level of 1 spore per 50 g sample with a 99% confidence). Pour the 50 g subsample of grains into a 250-mL Erlenmeyer flask (Web Fig. 8). Add 100 mL of 0.01% Tween-20 aqueous solution to the flask. Seal the top of the flask (e.g. with Parafilm or clingfilm). Place the flask on a flask shaker set at an appropriate speed (e.g. 350 oscillations min−1, or 200 rev min−1 for an orbital shaker) to ensure good agitation for 3 min to release any teliospores from the grain. Alternatively the flask can be shaken or swirled by hand. Place a 53-µm mesh nylon sieve (11 cm diameter) in a funnel over a clean 500-mL Erlenmeyer flask (Web Fig. 9) then pour the whole contents of the flask (the grain and the wash water) into the sieve (Web Fig. 10). Rinse the 250-mL flask with 20–50 mL of distilled water from an aspirator bottle, then pour evenly over the grain on the 53-µm sieve. Repeat this rinse twice more. Thoroughly rinse the grain on the 53-µm sieve by washing with further distilled water from an aspirator bottle (Web Fig. 10) to give a final collected volume of 300–400 mL. Remove the 53-µm sieve from the funnel and rinse the funnel with two aliquots of 10–20 mL of distilled water, collecting the water in the same 500 mL flask. (NB. Keep the washed grain sample(s) and also the remainder of the submitted sample that has not been tested, in case there is a need to examine grain directly for disease symptoms – see Appendix 3). Place a 20-µm mesh nylon sieve (4 cm diameter; Web Fig. 9) in a funnel over a second 500-mL Erlenmeyer flask. Pour the collected washings from the earlier washings through the 20-µm nylon sieve. (NB. Wet the sieve membrane prior to use and gently tap the outside of the PVC sieve-holder repeatedly to
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