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Diagnostic protocols for regulated pests Protocoles de diagnostic pour les organismes réglementés

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155

156 Diagnostic protocols

Approval ¥ laboratory procedures presented in the protocols may be adjusted to the standards of individual laboratories, provided EPPO Standards are approved by EPPO Council. The date of that they are adequately validated or that proper positive and approval appears in each individual standard. In the terms of negative controls are included. Article II of the IPPC, EPPO Standards are Regional Standards for the members of EPPO. References Review EPPO/CABI (1996) Quarantine Pests for Europe, 2nd edn. CAB Interna- tional, Wallingford (GB). EPPO Standards are subject to periodic review and amendment. EU (2000) Council Directive 2000/29/EC of 8 May 2000 on protective The next review date for this EPPO Standard is decided by the measures against the introduction into the Community of organisms EPPO Working Party on Phytosanitary Regulations harmful to plants or plant products and against their spread within the Community. Official Journal of the European Communities L169, 1Ð112. FAO (1997) International Plant Protection Convention (new revised text). Amendment record FAO, Rome (IT). IPPC (1993) Principles of plant quarantine as related to international trade. Amendments will be issued as necessary, numbered and dated. ISPM no. 1. IPPC Secretariat, FAO, Rome (IT). The dates of amendment appear in each individual standard (as IPPC (2002) Glossary of phytosanitary terms. ISPM no. 5. IPPC Secretariat, appropriate). FAO, Rome (IT). OEPP/EPPO (2003) EPPO Standards PM 1/2 (12): EPPO A1 and A2 lists of quarantine pests. EPPO Standards PM1 General phytosanitary meas- Distribution ures, 5Ð17. OEPP/EPPO, Paris. EPPO Standards are distributed by the EPPO Secretariat to all EPPO member governments. Copies are available to any Definitions interested person under particular conditions upon request to Regulated pest: a quarantine pest or regulated non-quarantine pest. the EPPO Secretariat. Quarantine pest: a pest of potential economic importance to the area endangered thereby and not yet present there, or present but not widely distributed and being officially controlled. Scope EPPO Diagnostic Protocols for Regulated Pests are intended to be used by National Plant Protection Organizations, in their Outline of requirements capacity as bodies responsible for the application of phyto- EPPO Diagnostic Protocols for Regulated Pests provide all the sanitary measures to detect and identify the regulated pests of information necessary for a named pest to be detected and the EPPO and/or European Union lists. positively identified by an expert (i.e. a specialist in In 1998, EPPO started a new programme to prepare diagnostic entomologist, mycology, virology, bacteriology, etc.). Each protocols for the regulated pests of the EPPO region (including the protocol begins with some short general information on the pest EU). The work is conducted by the EPPO Panel on Diagnostics (its appearance, relationship with other organisms, host range, and other specialist Panels. The objective of the programme is to effects on host, geographical distribution and its identity) and develop an internationally agreed diagnostic protocol for each then gives details on the detection, identification, comparison regulated pest. The protocols are based on the many years of experi- with similar species, requirements for a positive diagnosis, list ence of EPPO experts. The first drafts are prepared by an assigned of institutes or individuals where further information on that expert author(s). They are written according to a ‘common format organism can be obtained, references (on the diagnosis, and content of a diagnostic protocol’ agreed by the Panel on Dia- detection/extraction method, test methods). gnostics, modified as necessary to fit individual pests. As a general rule, the protocol recommends a particular means of detection or Existing EPPO Standards in this series identification which is considered to have advantages (of reliabil- ity, ease of use, etc.) over other methods. Other methods may also Nineteen EPPO standards on diagnostic protocols have already be mentioned, giving their advantages/disadvantages. If a method been approved and published. Each standard is numbered in the not mentioned in the protocol is used, it should be justified. style PM 7/4 (1), meaning an EPPO Standard on Phytosanitary The following general provisions apply to all diagnostic Measures (PM), in series no. 7 (Diagnostic Protocols), in this protocols: case standard no. 4, first version. The existing standards are: ¥ laboratory tests may involve the use of chemicals or appar- PM 7/1 (1) Ceratocystis fagacearum. Bulletin OEPP/EPPO atus which present a certain hazard. In all cases, local safety Bulletin 31, 41Ð44 procedures should be strictly followed PM 7/2 (1) Tobacco ringspot nepovirus. Bulletin OEPP/EPPO ¥ use of names of chemicals or equipment in these EPPO Bulletin 31, 45Ð51 Standards implies no approval of them to the exclusion of PM 7/3 (1) Thrips palmi. Bulletin OEPP/EPPO Bulletin 31, others that may also be suitable 53Ð60

EPPO Standards 157

PM 7/4 (1) Bursaphelenchus xylophilus. Bulletin OEPP/ PM 7/16 (1) Fusarium oxysporum f. sp. albedinis. Bulletin EPPO Bulletin 31, 61Ð69 OEPP/EPPO Bulletin 33, 265Ð270 PM 7/5 (1) Nacobbus aberrans. Bulletin OEPP/EPPO Bulletin PM 7/17 (1) Guignardia citricarpa. Bulletin OEPP/EPPO 31, 71Ð77 Bulletin 33, 271Ð280 PM 7/6 (1) Chrysanthemum stunt pospiviroid. Bulletin OEPP/ PM 7/18 (1) Monilinia fructicola. Bulletin OEPP/EPPO EPPO Bulletin 32, 245Ð253 Bulletin 33, 281Ð288 PM 7/7 (1) Aleurocanthus spiniferus. Bulletin OEPP/EPPO PM 7/19 (1) Helicoverpa armigera. Bulletin OEPP/EPPO Bulletin 32, 255Ð259 Bulletin 33, 289Ð296 PM 7/8 (1) Aleurocanthus woglumi. Bulletin OEPP/EPPO Several of the Standards of the present set result from a differ- Bulletin 32, 261Ð265 ent drafting and consultation procedure. They are the output PM 7/9 (1) Cacoecimorpha pronubana. Bulletin OEPP/EPPO of the DIAGPRO Project of the Commission of the European Bulletin 32, 267Ð275 Union (no. SMT 4-CT98-2252). This project involved four PM 7/10 (1) Cacyreus marshalli. Bulletin OEPP/EPPO ‘contractor’ diagnostic laboratories (in England, Netherlands, Bulletin 32, 277Ð279 Scotland, Spain) and 50 ‘intercomparison’ laboratories in many PM 7/11 (1) Frankliniella occidentalis. Bulletin OEPP/EPPO European countries (within and outside the European Union), Bulletin 32, 281Ð292 which were involved in ring-testing the draft protocols. The PM 7/12 (1) Parasaissetia nigra. Bulletin OEPP/EPPO DIAGPRO project was set up in full knowledge of the parallel Bulletin 32, 293Ð298 activity of the EPPO Working Party on Phytosanitary PM 7/13 (1) Trogoderma granarium. Bulletin OEPP/EPPO Regulations in drafting diagnostic protocols, and covered Bulletin 32, 299Ð310 regulated pests which were for that reason not included in the PM 7/14 (1) Ceratocystis ﬁmbriata f. sp. platani. Bulletin EPPO programme. The DIAGPRO protocols have been OEPP/EPPO Bulletin 33, 249Ð256 approved by the Council of EPPO as EPPO Standards in series PM 7/15 (1) Ciborinia camelliae. Bulletin OEPP/EPPO PM7. They will in future be subject to review by EPPO Bulletin 33, 257Ð264 procedures, on the same terms as other members of the series.

EuropeanBlackwell Publishing, Ltd. and Mediterranean Plant Protection Organization PM 7/29(1) Organisation Européenne et Méditerranéenne pour la Protection des Plantes

Diagnostic protocols for regulated pests1 Protocoles de diagnostic pour les organismes réglementés

Tilletia indica

Speciﬁc scope Speciﬁc approval and amendment This standard describes a diagnostic protocol for Tilletia indica. 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 common, but infection may also spread down the adaxial groove and, in severe cases, the whole grain may appear bunted Tilletia indica causes the disease Karnal bunt, or partial bunt, of (Web Fig. 1). 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) Sampling as a I/AI pest in 1996 and phytosanitary requirements applied to Seed lots should be sampled according to current ISTA rules. seed and grain of Triticum, Secale and X Triticosecale imported Grain, e.g. for feed or processing, is typically more difficult from countries where T. indica is known to occur. to sample because consignments are usually very large, and transported or stored as large, loose bulks. However, for Identity monitoring purposes, grain should be sampled in an appropriate fashion to produce a 1Ð2 kg thoroughly mixed sample repres- Name: Tilletia indica Mitra. entative of the consignment. Synonyms: Neovossia indica (Mitra) Mundkur. For phytosanitary purposes, detection of T. indica is best Taxonomic position: Fungi: Basidiomycota: Ustilaginales. achieved by a wash test (CABI/EPPO, 1997); infected parts Bayer computer code: NEOVIN. of the grain typically disintegrate so that the teliospores Phytosanitary categorization: EPPO A1 list, no. 23; EU contaminate other grains in the lot. The most efficient and rapid Annex designation I/AI. wash test method for detecting teliospores in a sample is a size- selective sieving and centrifugation technique (Appendix 1; Detection Peterson et al., 2000). This method has, on average, an 82% efficiency of recovery and microscopic examinations typically Symptoms require only a few slides per 50 g subsample. The number of replicate 50 g subsamples needed to detect differing levels of T. indica is a floret-infecting fungal smut pathogen. Unlike contamination is given in Table 1. systemic smuts, not all the seeds on an ear are usually infected. Direct visual examinations for bunted kernels or teliospores Seeds are infected through the germinal end of the grain and contaminating seed surfaces are not considered reliable methods the fungus develops within the pericarp where it produces a for phytosanitary purposes. However, Karnal bunt may be detected powdery, brownish-black mass of teliospores. When fresh, by visual examination with the naked eye and low power micro- the spore masses produce a foetid, decaying fish-like smell scopy (× 10Ð× 70 magnification). To help visualize symptoms, (trimethylamine). Seeds are usually only partially colonized, seed can be soaked in 0.2% NaOH for 24 h at 20 °C. This is showing various degrees of infection. Point infections are most especially useful for chemically treated seed lots where coloured dyes may obscure symptoms (Mathur & Cunfer, 1993; Agarwal 1The Figures in this Standard marked ‘Web Fig.’ are published on the EPPO & Mathur, 1992). With severe contamination, teliospores may website www.eppo.org. be seen on the surface of seeds (Mathur & Cunfer, 1993).

220 Tilletia indica

Table 1 Number of replicate 50 g subsamples needed to detect differing seeds or grain of wheat by a size-selective sieving wash test; levels of contamination with specified confidences, assuming an equal morphological identification of teliospores detected in wash distribution of teliospores (Peterson et al., 2000; Inman & Bowyer, EU tests; isolation and germination of teliospores for molecular SMT4-CT98-2252 evaluation, 2000) confirmation and molecular confirmation of cultures.

No. of replicate samples required for detection according to level of Confirmation confidence (%) Morphological confirmation Contamination level (no. of 99% 99.9% 99.99% If only a few teliospores are present (< 10 teliospores) in a wash spores per 50 g sample) test, morphological characters are not considered totally 1356reliable for confident discrimination between T. indica and the 2234morphologically similar species that are known contaminants 5111of wheat grain, e.g. T. walkeri and T. horrida (Table 2; Web Figs 3Ð5). However, if large numbers of teliospores are present (> 10 spores) then morphological identification may be possible Identification (Appendix 3). The most important discriminatory characters are teliospore size (maximum size and mean), exospore ornamenta- Isolation tion and colour (Table 2; Web Figs 3Ð5; Appendix 3). However, molecular confirmation tests are still recommended. T. indica is a facultative biotroph. To produce cultures, teliospores When suspect teliospores are found in a wash test, the grains are soaked in water, quickly surface-sterilized and then germinated in both the washed subsample(s) and the larger submitted on water-agar plates (Appendix 4). After 7Ð14 days, non- sample should be examined for Karnal bunt symptoms. If dormant teliospores produce a promycelium bearing 32Ð128 or symptoms are found, these should be confirmed by microscopic more basidiospores (primary sporidia) at its tip. These basidios- examination of the teliospores. Any grass seeds found in pores can then be cultured directly on solid or liquid nutrient media. the sample should also be examined for signs of bunt infection and, if found, the associated teliospores should be Morphology examined microscopically. Molecular confirmation tests are also recommended. Teliospores globose to subglobose, sometimes with a small hyphal fragment (more common on immature teliospores, Molecular confirmation but occasionally on mature teliospores), mostly 22Ð47 µm in There are three main molecular methods available to confirm diameter, occasionally larger (mean 35Ð 41 µm); pale orange to presumptive morphological diagnoses: (1) restriction enzyme brown to dark, reddish brown; some teliospores black and opaque analysis of the ITS1 region (rDNA Internally Transcribed (Web Fig. 3); densely ornamented with sharply pointed to Spacer Region 1) after PCR application using universal ITS truncate spines, occasionally with curved tips, 1.5Ð5.0 µm high, primers; (2) conventional PCR (polymerase chain reaction) assay which in surface view appear as either individual spines using species specific primers; (3) PCR assay using species (densely echinulate) or as closely spaced, narrow ridges (finely specific primers and a fluorescent probe in a TaqMan system. cerebriform) (Web Fig. 3); the spines are covered by a thin hyaline All these molecular confirmation methods require that membrane. Sterile cells: globose, subglobose to lacrymiform teliospores are germinated and cultures produced from the (tear-shaped), yellowish brown, 10Ð28 × 48 µm, with or without resulting sporidia (Appendix 4) Detailed descriptions of an apiculus (short stalk), with smooth walls up to 7 µm thick and these methods are still in preparation. Although PCR has been laminated. Sterile cells are likely to be uncommon in sieved wash- used on ungerminated teliospores, this typically requires large ings. See also CMI (1983), Table 2, Web Fig. 3, Appendix (3)2. numbers of teliospores and even then negative results are considered unreliable (Smith et al., 1996; McDonald et al., 1999). Diagnostic scheme The diagnostic scheme for T. indica, as presented in Fig. 2, Possible confusion with similar species describes procedures for detection of teliospores in imported Morphological comparisons

2The mounting medium, and heating or warming treatments, can affect Other tuberculate-spored Tilletia species may be confused with teliospore size (Aggarwal et al., 1990; Khanna & Payak, 1968; Castlebury T. indica (Durán & Fischer, 1961; Durán, 1987). In particularly, & Carris, 1999). This protocol assumes that spores are mounted in water the morphologically and genetically similar fungus Tilletia and not warmed or heated; suspect spores can then be germinated for walkeri (ryegrass bunt), and also Tilletia horrida (rice smut), any subsequent PCR conﬁrmation. However, surface ornamentation can sometimes not be seen clearly in water. In such cases, mounting teliospores are known contaminants of wheat seed or grain (Cunfer & in lactoglycerol or Shears’s solution (Mathur & Cunfer, 1993) and gently Castlebury, 1999; Castlebury & Carris, 1999; Smith et al., heating the slides may improve clarity. 1996). The most important morphological characters that

Diagnostic protocols 221

Fig. 2 Decision scheme for the detection and identiﬁcation of Tilletia indica. discriminate T. indica, T. walkeri and T. horrida are teliospore size Web Figs 3Ð5; Appendix 3). If sufﬁcient numbers of telio- (range and mean), exospore ornamentation and colour (Table 23; spores 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 3The literature on spore sizes is often variable. Spore size is affected by the that appear as polygonal scales in surface view. T. walkeri and mounting medium and by heating treatments. For rice smut (T. horrida, T. indica have a larger degree of overlap in morphological synonym T. barclayana), data from rice is potentially more reliable than data characters. However, T. walkeri teliospores are on average based on T. barclayana sensu lato from various Poaceae as the latter is considered a species complex. The rice pathogen is considered distinct smaller, paler in colour (never black/opaque) and have coarser from those on Paspalum and Panicum, but it is not known whether it is distinct exospore ornamentation which in surface view gives the from T. barclayana s.s. on Pennisetum (Pimentel, 1998; Castlebury, 1998). appearance of wide, incompletely cerebriform ridges or thick

222 Tilletia indica

Table 2 Morphological characteristics of Tilletia indica, Tilletia walkeri and Tilletia horrida

Teliospore character Tilletia indica1 Tilletia walkeri2 Tilletia horrida3

Size (range) µm22Ð47Ð(61)(26Ð55(−64)) (23Ð45) 17Ð36(20Ð38(−41)) Size (mean) µm35Ð41 30Ð31* 24Ð28‡ (40Ð44)† (34Ð36)† (28)† Colour Pale orange to mainly dark, Pale yellow to mainly dark Pale yellow to mainly light or reddish brown to opaque-black reddish brown (never opaque) dark chestnut brown (semiopaque) Exospore Sharply pointed to truncate Conical to truncate spines Sharply pointed or curved spines, ornamentationin median spines (occasionally curved), (occasionally curved), 1.5Ð4.0 µm high, becoming truncated view (Hawksworth et al., 1.5Ð5.0 µm high, covered with 3Ð6 µm high, covered with a scales with maturity, covered with 1995) a hyaline sheath hyaline to yellowish-brown sheath a hyaline to tinted sheath‡ Exospore ornamentation Spines densely arranged, either Spines coarsely arranged, forming Spines appearing as polygonal in surface view individually (densely echinulate) wide, incompletely cerebriform scales (occasionally spines forming or in closely spaced, narrow (to coralloid) ridges or thick clumps cerebriform ridges or small clumps) ridges (ﬁnely cerebriform)

1Authors’ data. 2Based on: Castlebury & Carris (1999); Cunfer & Castlebury (1999); Milbrath et al. (1998); Castlebury (1998). 3As 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).

clumps (Castlebury & Carris, 1999). In median view, the Molecular comparisons exospore spine profiles may also aid identification. The median profiles can be enhanced by bleaching the teliospores in 10% Diagnostically significant differences exist between T. indica, sodium hypochlorite for 15Ð20 min. If necessary, spores T. walkeri and T. horrida in their nuclear and mitochondrial can then be rinsed twice in water and stained, e.g. with trypan DNA. Interspecific polymorphisms have been identified using blue or cotton blue in lactoglycerol (Web Fig. 6). In general, various polymerase chain reaction (PCR) methods, including T. indica teliospores have a smoother, more complete median RAPDs, RFLPs and AFLPs (Pimentel et al., 1998; Laroche outline due to their spines being more densely arranged; profiles et al., 1998). In the nuclear ribosomal (rDNA) ITS1 and ITS2 of T. walkeri are more irregular with gaps between the spines regions, there is a > 98% similarity between T. walkeri and due their spines being more coarsely arranged (Web Fig. 6). T. indica sequences (Levy et al., 1998). However, within the In culture, T. walkeri and T. indica produce very similar ITS1 region, T. walkeri has a diagnostically important restriction colonies. On potato dextrose agar (PDA) after 14 days at 19 °C enzyme site (ScaI) that is not present with T. indica, T. horrida with a 12-h light cycle, both species typically produce white or other closely related species (Pimentel et al., 1998; to cream-coloured, slow-growing, irregular, crustose colonies, Levy et al., 1998). With mtDNA, sequence differences have about 4Ð6 mm in diameter (Web Fig. 7). In comparison, enabled species-specific primers to be designed to T. indica comparable cultures of T. horrida grow significantly more and T. walkeri (Frederick et al., 2000). These primers can slowly (colonies only 2Ð3 mm in diameter) due to their higher be used in conventional PCR assays or in a TaqMan system temperature optima. T. horrida isolates may also produce a in conjunction with a probe (Frederick et al., 2000). There reddish-purple pigment (Web Fig. 7), both on PDA and potato are currently no species-specific primers for T. horrida, but dextrose broth. RFLPs can be used to identify cultures (Pimentel et al., 1998). Other tuberculate-spored Tilletia species have teliospores If species-specific primers for T. walkeri and T. indica do that can appear morphologically similar to those of T. indica not give positive results on test cultures, RFLPs, RAPDs or (Pimentel et al., 1998; Durán & Fischer, 1961). These species AFLPs may be useful tools in identification (Pimental et al., are less likely to be found as contaminants of wheat, but they 1998). include: Tilletia barclayana (smut of various Poaceae, e.g. Panicum and Paspalum), Tilletia eragrostidis (on Eragrostis), Requirements for a positive diagnosis Tilletia inolens (on Lachnagrostis filiformis), Tilletia rugispora (on Paspalum), Tilletia boutelouae (on Bouteloua gracilis). The procedures for detection and identification described in this None of these morphologically similar species, or T. walkeri or protocol, and the decision scheme in Fig. 2, should have been T. horrida, has been found naturally to infect wheat. followed.

If bunted grains are present, a positive diagnosis can be made Further information if the symptoms are confirmed by the presence of teliospores that are morphologically consistent with those described for Further information on this organism can be obtained T. indica in this protocol (Table 2; Appendix 3). from: Pest and Disease Identification Team, Central Science If bunted grains are not present, the wash test and procedures Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom. for detection described in this protocol should be followed. Since there is considerable overlap in morphological characters Acknowledgements between T. indica and various other tuberculate-spored species which can potentially contaminate seed or grain, This protocol was originally drafted by: A. J. Inman, K. J. D. namely T. walkeri and T. horrida, morphological identifi- Hughes and R. J. Bowyer, Central Science Laboratory, York cation of teliospores is only possible when a large number (GB). of teliospores are present (> 10 spores). In such cases, This protocol was ring-tested in different European teliospores may be identified morphologically, but only if laboratories4. all key characters conform to one specific species (size range, size mean, colour and exospore ornamentation patterns: (see Appendix 3). Molecular confirmation is still References recommended. Agarwal R, Joshi LM & Singh DV (1990) Morphological differences If too few spores are present (< 10 spores), it may not be between teliospores of Neovossia indica and N. horrida. Indian Phytopa- possible to discriminate species using morphological thology 43, 439Ð442. characters. In such cases, molecular confirmation by PCR Agarwal VK & Mathur SB (1992) Detection of Karnal bunt in wheat seed using species – specific primers combined as appropriate with samples treated with fungicides. FAO Plant Protection Bulletin 40, 148Ð restriction Ð enzyme analysis is recommended for a positive 153. diagnosis. Bonde MR, Nester SE, Smilanick JL, Frederick RD & Schaad NW (1999) Comparison of effects of acidic electrolysed water and NaOCl on Tilletia indica teliospore germination. Plant Disease 83, 627Ð632. Report on the diagnosis Castlebury LA (1998) Morphological characterisation of Tilletia indica and similar fungi. In: Bunts and Smuts of Wheat: an International A report on the diagnosis should include the following Symposium (Ed. Malik VS & Mathur DE), pp. 97Ð105. NAPPO, Ottawa information: (CA). • results obtained by the recommended procedures Castlebury LA & Carris LM (1999) Tilletia walkeri, a new species on • the origin (country; state) and type (host; commodity) of the Lolium multiflorum and L. perenne. Mycologia 91, 121Ð131. CMI (1965) Description of Pathogenic Fungi and Bacteria, no. 75. Tilletia infected material barclayana. CAB International, Wallingford (GB). • quantity of consignment or lot CMI (1983) Description of Pathogenic Fungi and Bacteria, no. 748. Tilletia • degree of infection/contamination (e.g. the number of indica. CAB International, Wallingford (GB). positive subsamples and the estimated number of teliospores Cunfer BM & Castlebury LA (1999) Tilletia walkeri on annual ryegrass detected in each positive subsample) in wheat fields in the southeastern United States. Plant Disease 83, 685Ð689. • a description of any disease symptoms (preferably including Durán R (1987) Ustilaginales of Mexico: Taxonomy, Symptomatology, Spore Germination and Basidial Cytology. Washington. State University, photographs) Seattle (US). • a description of the teliospores (with colour photographs Durán R & Fischer GW (1961) The Genus Tilletia. Washington. State if possible) compared with descriptions of T. indica and University, Seattle (US). morphologically similar species, e.g. T. walkeri and T. EPPO/CABI (1997) Quarantine Pests for Europe, 2nd edn. CAB Interna- horrida tional, Wallingford (GB). • if cultures were obtained for molecular confirmation, a Frederick RD, Snyder KE, Tooley PW, Berthier-Schaad Y, Peterson GL, description of the colony morphology, especially any Bonde MR, Schaad NW & Knorr DA (2000) Identification and differen- tiation of Tilletia indica and T. walkeri using the polymerase chain pigmentation, and growth rate under defined conditions reaction. Phytopathology 90, 951Ð960. Cultures should be kept (mycelium from broths or mycelial Hawksworth DL, Kirk PM, Sutton BC & Pegler DN (1995) Ainsworth and plugs from agar plates can be stored frozen at −80 °C) Bisby’s Dictionary of the Fungi, 8th edn. CAB International, Wallingford • if molecular confirmation tests were done, a copy of the (GB). molecular test result along with positive and negative controls should be provided. For TaqMan assays, the CT-value should 4A. Radova, State Phytosanitary Administration, Olomouc, CZ; I. be noted Vloutoglou, Benaki Phytopathological Institute, Athens (GR); A. Porta- • comments as appropriate on the certainty or uncertainty of Puglia, Istituto Sperimentale per la Patologia Vegetale, Rome (IT); C. the identification. Montuschi, Servizio Fitosanitario Regionale, Bologna (IT); I. van Browershaven, Plantenziektenkundige Dienst, Wageningen (NL); M. de When cultures have been obtained, these should be cultured Jesus Gomes, E. Diogo & M.R. Malheiros, Direcção-Geral de Protecção das onto potato dextrose agar (PDA) and 5-mm diameter mycelial Culturas, Lisboa (PT); V. Cockerell, Scottish Agricultural Science Agency, plugs stored at −80 °C in sterile tubes. Similarly, any DNA East Craigs, Edinburgh (GB); A. Barnes, Central Science Laboratory, Sand extractions should be stored at −80 °C. Hutton, York (GB).

Khanna A & Payak MM (1968) Teliospore morphology of some smut fungi. teliospores after a few minutes exposure and little germination II. Light microscopy. Mycologia 60, 655Ð662. can be expected after exposure of 1 h). Laroche A, Gaudet DA, Despins T, Lee A & Kristjansson G (1998) Distinction between strains of Karnal bunt and grass bunt using amplified fragment length polymorphism (AFLP). In: Bunts and Smuts of Wheat: Method an International Symposium (Ed. Malik VS & Mathur DE), p. 127. NAPPO, Ottawa (CA). Bleach the sieves, funnels and flasks by immersion for 15 min Levy L, Meyer RJ, Carris L, Peterson G & Tschanz AT (1998) Differentia- in 30% bleach6. Rinse the bleach thoroughly from the equipment tion of Tilletia indica from the undescribed Tilletia species on ryegrass by with tap water. Weigh 50 g of grain into a new, disposable, its sequence differences. Proceedings of the 12th Biennial Workshop on large weigh boat (see Fig. 2, Table 1, for the number of 50 g Smut Fungi, p. 29. subsamples required to detect different levels of contamination; Mathur SB & Cunfer BM (1993) Seed-Borne Diseases and Seed Health Testing of Wheat, pp. 31Ð43. Danish. Government Institute of Seed 3 replicates detects a level of 1 spore per 50 g sample with a Pathology for Developing Countries, Copenhagen (DK). 99% confidence). Pour the 50 g subsample of grains into a McDonald JG, Wong E, Kristjansson GT & White GP (1999) Direct 250-mL Erlenmeyer flask (Web Fig. 8). Add 100 mL of 0.01% amplification of PCR of DNA from ungerminated teliospores of Tilletia Tween-20 aqueous solution to the flask. Seal the top of the flask species. Canadian Journal of Plant Pathology 21, 78Ð80. (e.g. with Parafilm or clingfilm). Milbrath GM, Pakdel R & Hilburn D (1998) Karnal bunt spores in ryegrass Place the flask on a flask shaker set at an appropriate speed (Lolium spp.). In: Bunts and Smuts of Wheat: an International Symposium (e.g. 350 oscillations min−1, or 200 rev min−1 for an orbital shaker) (Ed. Malik, VS & Mathur, DE), pp. 113Ð116. NAPPO, Ottawa (CA). NAPPO (1999) NAPPO Standards for Phytosanitary Measures: a harmo- to ensure good agitation for 3 min to release any teliospores nized procedure for morphologically distinguishing teliospores of Karnal from the grain. Alternatively the flask can be shaken or swirled bunt, ryegrass bunt and rice bunt. http://www.nappo.org. by hand. Place a 53-µm mesh nylon sieve (11 cm diameter) in Peterson GL, Bonde MR & Phillips JG (2000) Size-selective sieving for a funnel over a clean 500-mL Erlenmeyer flask (Web Fig. 9) detecting teliospores of Tilletia indica in wheat seed samples. Plant then pour the whole contents of the flask (the grain and the Disease 84, 999Ð1007. wash water) into the sieve (Web Fig. 10). Rinse the 250-mL Pimentel G, Carris LM, Levy L & Meyer R (1998) Genetic variability flask with 20Ð50 mL of distilled water from an aspirator bottle, among isolates of Tilletia barclayana, T. indica and allied species. µ Mycologia 90, 1017Ð1027. then pour evenly over the grain on the 53- m sieve. Repeat this Smith OP, Peterson GL, Beck RJ, Schaad NW & Bonde MR (1996) rinse twice more. Thoroughly rinse the grain on the 53-µm Development of a PCR-based method for identification of Tilletia indica. sieve by washing with further distilled water from an aspirator Phytopathology 86, 115Ð122. bottle (Web Fig. 10) to give a final collected volume of 300Ð400 mL. Remove the 53-µm sieve from the funnel and rinse the funnel Appendix 1. Method for extracting teliospores with two aliquots of 10Ð20 mL of distilled water, collecting the from untreated seed or grain by size-selective water in the same 500 mL flask. (NB. Keep the washed grain sieving (based on Peterson et al., 2000) sample(s) and also the remainder of the submitted sample that has not been tested, in case there is a need to examine grain Materials directly for disease symptoms Ð see Appendix 3). Place a 20-µm 30% bleach solution (3 parts household bleach: 7 parts water; mesh nylon sieve (4 cm diameter; Web Fig. 9) in a funnel over c. 1.6% active NaOCl); wash water = 0.01% aqueous Tween-20 a second 500-mL Erlenmeyer flask. Pour the collected (detergent); large weigh boats (8 × 8 cm); weighing balance; washings from the earlier washings through the 20-µm nylon 250-mL Erlenmeyer glass flask; 100-mL measuring cylinder; sieve. (NB. Wet the sieve membrane prior to use and gently tap Parafilm’M′ or clingfilm; laboratory flask shaker (alternatively, the outside of the PVC sieve-holder repeatedly to facilitate a shaking flasks by hand is acceptable); 500-mL Erlenmeyer good rate of sieving; otherwise the membrane can quickly glass flask × 2; funnel (approx. 13 cm diameter); 53 (50 or 70) become blocked.) µm mesh nylon sieve (11 cm external diameter) mesh from Rinse the first 500-mL flask twice with 20 mL of water and Spectrum Laboratories; 20-µm mesh nylon sieve (4 cm external pour through the 20-µm sieve. Tilt the 20-µm sieve to an angle diameter): mesh from BDH or Spectrum Laboratories; aspirator of 30Ð45° (Web Fig. 11) and gently wash the deposit on the bottle with distilled water; sterile disposable 3 mL Pasteur µ pipettes; pipettor (100- L capacity) plus disposable pipettor 5 Shear’s solution: 300 mL Mc Ilvaine’s buffer, 6 mL Potassium acetate, tips (plugged); pipettor (1000-µL capacity) plus disposable 120 mL Glycerine 180 mL Ethyl alcohol (95%). Prepare Mc Ilvaine’s buffer pipettor tips (plugged); 15-mL sterile, disposable conical (Mathur & Cunfer, 1993) as follows: dissolve 19.212 g citric acid in bottom centrifuge tubes; centrifuge (to take 15-mL centrifuge 1000 mL distilled water and mix thoroughly; dissolve 28.392 g of disodium tubes above); autoclavable, disposable waste bottle; autoclave phosphate (Na2HPO4) in 1000 mL of distilled water and mix thoroughly; bags; glass microscope slides (76 × 21 mm); microscope mix 8.25 mL of citric acid solution with 291.75 mL of disodium phosphate solution and mix thoroughly. cover slips (18 × 18 mm); compound microscope (× 100Ð 400 6Bleach eliminates the risk of false positives by cross contamination from × magnification); dissecting microscope ( 10Ð70 magnification); previous samples; bleach kills teliospores and makes them appear hyaline Shear’s solution5 (as an alternative mounting medium to water compared with the normally dark, pigmented spores. The bleach solution if slides are prone to drying; however, Shear’s starts to kill should be changed regularly, as appropriate.

membrane and on the sieve walls on to one side of the Table 3 Example of a record sheet, with suggested colour and membrane using distilled water or 0.01% Tween 20 detergent ornamentation codes, that could be used for teliospores detected in wash from an aspirator bottle or a disposable Pasteur pipette. Recover tests the suspension that collects at the edge of the 20-µm sieve using Teliospore Size (µm) a clean, disposable Pasteur pipette (Web Fig. 12) and place the number (diam.) Colour Ornamentation Notes suspension in a new 15-mL disposable conical centrifuge tube. Repeat these steps until the 20-µm sieve appears clean (this may 1 require 5–10 repeats, and typically results in a final collected 2 volume of about 3Ð5 mL in the centrifuge tube). If necessary, 3 4 the 20-µm sieve can be examined under a low power micro- 5 scope to check for any residual teliospores. 6 Centrifuge the collected suspension at 1000 g for 3 min7. 7 Carefully remove the supernatant using a 1-mL pipettor with a etc. plugged, disposable pipette tip, or a new disposable Pasteur Size range Mean ± s.d. Provisional identification pipette. Take care not to disturb the pellet (discard the removed Examples of colour codes: BO black/opaque, RB reddish brown, CB supernatant into a disposable waste vessel for quarantine chestnut brown, P (PY, PO, PB) pale (yellow/orange/brown). disposal). Re-suspend the pellet using distilled water to give a final Examples of ornamentation codes: DE densely echinulate (spines densely volume of 50Ð100 µL, or more if the pellet volume requires8. and individually arranged); FC finely cerebriforn (spines forming closely Pipette a 20-µL aliquot of the suspension onto a microscope spaced narrow ridges), CC coralloid (ridges much branched), CO coarsely slide and place a cover slip (18 × 18 mm) on top. Examine the cerebriform (spines coarsely arranged forming wide, incompletely whole slide immediately (the slide can quickly dry out) for cerebriform ridges), TC thick clumps (spines forming thick clumps), PS polygonal scales (curved in profile). teliospores of T. indica (Web Fig. 13) using a compound microscope at × 100Ð× 400 magnification. Assess the characteristics of any teliospores at × 100 magnification. Teliospores of T. indica are mainly 25Ð45 µm in diameter, pale orange but (NaOH) and incubate for 24 h before adding 100 mL of 0.01% mostly reddish-brown to opaque-black, and densely echinulate. Tween-20 aqueous solution to the flask. NaOH can help to (For reference, see: Web Figs 2 and 3, Tables 2 and Appendix 3). remove most of the fungicide, allowing subsequent sieving. Repeat with further 20-µL aliquots until the whole suspension Without the NaOH treatment, the 20-µm sieve may become has been examined. blocked by fungicide. The NaOH treatment does not affect If suspect teliospores are found, refer to, and follow, the teliospore size or colour characteristics, but does kill the morphological diagnostic method (Appendix 3) and the teliospores (Bowyer & Inman, unpublished). An alternative to general Diagnostic Scheme (Fig. 2), i.e. record morphological using NaOH is to use just the 53-µm sieve and not the 20 µm characters (e.g. size, colour, ornamentation); examine the sieve, if this becomes easily blocked. This method has not sample for bunted seeds; isolate & germinate teliospores for been ring-tested. molecular confirmation, if required. Finally, bleach all equipment used and rinse with water Appendix 3. Method for morphological before re-using (see Steps 1 & 2). identification If tuberculate teliospores are found in a wash test, record the Appendix 2. Method for extracting teliospores morphological characteristics of the teliospores using Table 3 from fungicide-treated seed by size-selective (refer also to Web Figs 3, 4, 5 and 13 and Table 2)9. Follow the sieving (adapted from Agarwal & Mathur, 1992 Decision Scheme (Web Fig. 15) and examine the submitted and based on Peterson et al., 2000) grain sample (including the 50 g washed subsamples) for Follow the Method in Appendix 1, but after weighing the 50-g bunted wheat seeds or bunted seeds of other Poaceae, e.g. sample add to it 100 mL of 0.2% (or 1%) sodium hydroxide ryegrass seed (see Fig. 2). If wheat seeds with Karnal bunt symptoms are found, confirm Tilletia indica by microscopic

7Conical-bottomed tubes are recommended, as are centrifuges with swing-out examination of the teliospores in the seed (Fig. 2, Table 2; arms rather than fixed arms, as these give better pellets. If debris is seen to Table 4). If bunted ryegrass seeds are found, but no bunted adhere to the inside walls of the centrifuge tubes, re-suspend in 0.01% wheat seeds, confirm T. walkeri by microscopic examination of Tween 20 and repeat the centrifugation the teliospores in the seed (Table 4). If confirmed, compare 8If 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 9Tuberculate teliospores detected in wash tests of wheat grain are assumed in Shear’s and little germination can be expected after exposure of 1 h. Slides to be either Tilletia indica, T. walkeri or T. horrida. Other tuberculate- should be assessed immediately (within 10Ð20 min) and any spores spored Tilletia species that infect various grasses cannot be excluded as recovered immediately from the slide (see Appendix 4) and washed in water contaminants, but have not previously been found contaminating wheat; see to allow germination and the recommended molecular confirmations Fig. 2

teliospores from the seed with those found in the wash test. If c the teliospores are identical make a diagnosis. Molecular conﬁrmation of the teliospores from the wash test is still b,c recommended. If wheat seeds infected with T. indica or ryegrass seeds infected with T. walkeri are not found, make a

Dense; echinulate or closely spaced ridges narrow (ﬁnely cerebriform) Sharpely pointed to truncate, occasionally curved or no gaps (few between spines after bleaching) presumptive identiﬁcation of teliospores found in the wash test: use Table 4, in conjunction with the following ‘guiding

c diagnostic principles’ (adapted from NAPPO, 1999): and median proﬁle b • 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 m sieves. m sieves. - character conforms to species µ

Coarse; broad, incompletely cerebriform ridges (to coralloid), or thick clumps. Conical to truncate (gaps between spines in profile obvious after bleaching) T. indica • samples with teliospores mostly 28Ð35 µm, translucent brown, never black/opaque, very spherical, with blunt spines m and 53 µ 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 Spines (ornamentation) in surface view Spines (ornamentation) in surface Echinulate; polygonal scales view; in surface occasionally cerebriform ridges or rarely clumps. Sharpely pointed, becoming truncate, occasionally curved • 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, semiopaque). ale orange, ut mostly P b dark reddish- to brown opaque black If relatively large numbers of teliospores are present (e.g. over 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 (Table 4). However, molecular confirmation is still recommended ale yellow ale yellow detected in size-selective sieving wash tests that use 20 wash sieving detected in size-selective P to mostly reddish-brown opaque) (never if bunted wheat seeds were not found in the grain sample. If only a few teliospores are detected (e.g. less than 10), or horrida morphological characters are not conclusive, then molecular T.

b methods are recommended for conﬁrmation of any presumptive and diagnosis (see Appendices IVÐVII). Colour to mostly light or dark chestnut- (to brown semi-opaque)

Appendix 4. Method for isolation and germination of teliospores for molecular a

m) conﬁrmation µ illetia indica, T. walkeri T. illetia indica, T Materials Aspirator bottle with distilled water; 20-µm mesh nylon sieve (4 cm diameter); sterile disposable Pasteur pipettes; Refer to Fig. 2 (Diagnostic Scheme) and Web Fig. 15 (Decision scheme). Fig. Web Fig. 2 (Diagnostic Scheme) and Refer to Mean size (diam., µ d pipettor (1000- L capacity) and sterile, disposable pipettor tips (plugged); pipettor (200-µL capacity) and sterile, disposable pipettor tips (plugged); sterile, disposable 3-mL Pasteur pipettes; eb Fig. 6; eb Fig. 15-mL disposable conical bottom centrifuge tubes; centrifuge W a c

m) (to take 15-mL centrifuge tubes); autoclavable disposable µ waste bottle; autoclave bags; 10% Bleach solution10 (1 part domestic bleach: 9 parts water; c. 0.3Ð0.5% active NaOCl); sterile distilled water; sterile disposable spreader; antibiotic Max Size (diam., < 36 36Ð45 45Ð50 + 24Ð28 30Ð31 35Ð41 yellow Pale

able 2 and Web Figs 3Ð5; Figs Web able 2 and 10 T Acidic electrolysed water (AEW) with the following properties can be b Scheme for morphologically distinguishing teliospores of

d used instead of 10% bleach (Bonde et al., 1999): pH 2.3Ð2.8; redox potential (ORP) c. 1100 mv; free chlorine (5)Ð10Ð15 (ppm). Equipment: boxes) able 2; able 2; horrida walkeri indica able 4 √ T T Sample ( T. T. T. a Super Oxseed Labo, Advanced H2O Inc., Alameda, US.

© 2004 OEPP/EPPO, Bulletin OEPP/EPPO Bulletin 34, 219 –227 Diagnostic protocols 227 water agar (AWA) plates (2% Technical agar No. 3, Oxoid; Centrifuge at 1200 g for 5 min, aseptically remove the 60 mg penicillin-G (Na salt) and 200 mg streptomycin sulphate supernatant and then add another 1 mL SDW to wash the pellet per L of agar); Parafilm M or electrical tape; scalpel; potato again. Centrifuge at 1200 g for 5 min, aseptically remove the dextrose broth (Difco); dissecting needle; sterile filter paper; supernatant and resuspend the final pellet in 1 mL SDW. 1.5Ð2.0 mL microcentrifuge tubes Transfer 200 µL of the teliospore suspension aseptically onto individual 2% water agar plates12 with antibiotics (AWA) and spread with a sterile spreader. Incubate the AWA plates at 21 °C Method with a 12-h light cycle (e.g. TLD 18 W/83 Philips white light Recover suspect teliospores from both the microscope slide tubes). Leave for about 5 days before sealing plates with and the cover slip by washing them with distilled water over a parafilm or electrical tape, or placing the plates inside clear clean 20-µm sieve. Recover the teliospores from the sieve polyethylene bags. After 7Ð14 days, examine the plates for (Appendix 1), pipetting the suspension into a new 15-mL germinated teliospores bearing a tuft of filiform basidiospores conical centrifuge tube. Make up the final volume to 3Ð5 mL (primary sporidia), or small colonies forming around germinated with distilled water. Incubate the teliospore suspension teliospores (Web Fig. 16). These colonies produce secondary overnight at 21 °C to hydrate the teliospores and make fungal sporidia typically of two types: filiform and allantoid. Cut out and bacterial contaminants more susceptible to subsequent small blocks of agar (5 × 5Ð10 × 10 mm square) bearing surface sterilization. After incubating overnight, pellet the germinated teliospores or colonies; invert the agar blocks and teliospores by centrifuging at 1200 g for 3 min. Remove the stick them onto the inside surface of a Petri dish lid. Place the supernatant with a pipettor with a plugged tip, or a disposable lids over Petri dish bases containing approximately 5 mL potato Pasteur pipette, taking care not to disturb the pellet; pipette dextrose broth so that sporidia can then be released onto the the supernatant into a suitable disposable waste bottle for broth surface. Incubate at 21 °C with 12 h light cycle. After 2Ð subsequent quarantine disposal. Resuspend the pellet in 5 mL 3 days, basidiospores deposited onto the broth surface produce of 10% domestic bleach11 (0.3Ð0.5% active NaOCl); replace small mats of mycelia approximately 0.5Ð1.0 cm diameter. the centrifuge tube cap and immediately invert the tube three With a sterile dissecting needle, remove each mycelial mat, time to ensure that the bleach contacts all inner surfaces. touching them onto sterile filter paper to remove excessive Immediately centrifuge at 1200 g for 1 min, then quickly and broth. Place the mycelia in suitable vials (e.g. 1.5Ð2.0 mL aseptically remove the supernatant and resuspend the pellet in microcentrifuge tubes) for immediate DNA extraction, or store 1 mL sterile distilled water (SDW) to wash the pellet. at −80 °C for subsequent DNA extraction.

11Instead of bleach, teliospores can be surface sterilized for 30 min in 5Ð 10 mL of AEW (see above). AEW effectively surface sterilizes teliospores 123-day-old plates are recommended, as these quickly absorb the but, compared to a 1Ð2 min bleach treatment, stimulates rather than reduces suspension; excessive surface water can inhibit teliospore germination. teliospore germination (Bonde et al., 1999). Alternatively, prepare the agar plates on the day of use, but pour the liquid NB. Some teliospores can be killed if the total time in the bleach exceeds 2 min agar when cool and do not replace the lids fully until the agar has set.

Fig. 1. Grain infected with Tilletia indica (Karnal bunt). Symptoms range from partial bunting (point infections and infections spreading down the adaxial groove) to almost complete bunting. Photograph courtesy of G. L. Peterson, USDA.

Fig. 3. Teliospores of Tilletia indica (Karnal bunt of wheat) showing surface ornamentation patterns: spines densely arranged, either individually (densely echinulate) or in closely spaced, narrow ridges (finely cerebriform). (Figures 3–5 are at the same scale; 10 mm = 17 µm.)

Fig. 4. Teliospores of Tilletia walkeri (ryegrass bunt) showing surface ornamentation patterns: spines coarsely arranged and forming wide, incompletely cerebriform to coralloid ridges or thick clumps. (Figures 3-5 are at the same scale; 10 mm = 17 µm.)

Fig. 5. Teliospores of Tilletia horrida (rice smut) showing surface ornamentation patterns: polygonal scales or, occasionally, with cerebriform ridges. (Figures 3–5 are at the same scale; 10 mm = 17 µm.)

Fig. 6. Teliospores of Tilletia indica (top) and T. walkeri (bottom) showing teliospore profiles in median view after bleaching and then staining with lactoglycerol-trypan blue. Note the smoother outline on T. indica teliospores compared to the more irregular outline of T. walkeri teliospores with more obvious gaps between spines.

Tilletia indica

Tilletia walkeri

Fig 7. Colonies of Tilletia indica (right), T. walkeri (centre) and T. horrida (left) after 7 days (top), 10 days (centre) and 14 days (bottom) on PDA at 19°C and a 12 hour dark/light cycle. Note slower growth, and purple pigmentation after 14 days, for T. horrida colonies. s y 7 da

s y 10 da

s y 14 da

T. horrida T. walkeri T. indica

Fig. 8 Size-selective sieving wash test: 20 µm mesh nylon sieve (mounted between 4 cm diameter cylinders: left), 53 µm sieve (mounted between 11 cm diameter cylinders; right) and a 50 g grain sample in a 250 ml Erlenmeyer flask with 100 ml of 0.01% Tween-20 aqueous solution.

Fig. 9 Size-selective sieving wash test: arrangement of sieves (20 µm, sieve, left; 53 µm sieve, right) mounted in funnels over 500 ml Erlenmeyer flasks in preparation for size-selective sieving of wash water from a 50 g grain sample.

Fig. 10 Size-selective sieving wash test: grain and washings poured onto a 53 µm sieve over a 500 ml Erlenmeyer flask, together with an aspirator bottle for subsequent rinsing of the grain on the sieve.

Fig. 11 Size-selective sieving wash test: the final sieve fraction being washed to one side of the 20 µm sieve with water from a disposable Pasteur pipette in preparation for recovery.

Fig. 12 Size-selective sieving wash test: the final sieve fraction being recovered from the 20 µm sieve with water from a disposable Pasteur pipette for subsequent centrifugation in a conical centrifuge tube.

Fig. 13. Tilletia indica teliospores in median view (20–50 µm diam.; mean 35–41 µm).

Fig. 15. Pictorial key to teliospore ornamentation (see also Figs 3–5).

Densely echinulate (DE)

Densely echinulate (DE) Tilletia indica to finely cerebriform

(FC: closely spaced, narrow ridges)

Coarsely cerebriform (CO): Coarsely arranged

spines forming wide incompletely cerebriform ridges

Incompletely cerebriform to coralloid

ridges (CC)

Tilletia walkeri

Thick clumps (TC)

Spines as polygonal scales (PS), sometimes

forming ridges or clumps (left and centre); Sharply pointed, often

Tilletia horrida curved spines (right)

Fig. 16. Tilletia indica teliospores germinating on water agar after 10–14 days, producing a tuft of primary sporidia (basidiospores) at the apex of the promycelium. Primary sporidia germinate in situ to produce small colonies which produce secondary sporidia of two types: further filiform sporidia; allantoid sporidia which are forcibly discharged.