Tilletia indica

Abstract

EPPO BulletinVolume 37, Issue 3 p. 503-520 Free Access Tilletia indica Correction(s) for this article Tilletia indica Volume 42Issue 3EPPO Bulletin pages: 512-512 First Published online: December 12, 2012 First published: 07 December 2007 https://doi.org/10.1111/j.1365-2338.2007.01158.xCitations: 4 European and Mediterranean Plant Protection Organization 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 onFacebookTwitterLinked InRedditWechat PM 7/29 (2) 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 (×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 an I /AI pest in 1996 and phytosanitary requirements are applied to seed and grain of Triticum, Secale and ×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: Ustilaginomycetes: Tilletiales 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 (Fig. 1). Figure 1Open in figure viewerPowerPoint 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. 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 (EPPO/CABI, 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, Hughes & Bowyer, 2003) Contamination level (no. of spores per 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 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 (Appendix 2). This is especially useful for chemically treated seed lots where coloured dyes may obscure symptoms (Agarwal & Mathur, 1992; Mathur & Cunfer, 1993). With severe contamination, teliospores may be seen on the surface of seeds (Mathur & Cunfer, 1993). Identification The flow diagram for the detection and identification of T. indica (Fig. 2), describes procedures for detection of teliospores in 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 Flow diagram for the detection and identification of T. indica. 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 by comparison of key characters e.g. size range, size mean, colour and exospore ornamentation patterns (see Appendix 3). 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 or combined as appropriate with restriction enzyme analysis is recommended requiring teliospore germination (Appendix 4). Table 2. Morphological characteristics of Tilletia indica, Tilletia walkeri and Tilletia horrida Teliospore character Tilletia indica a Tilletia walkeri b Tilletia horrida c 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 ornamentation in 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) a Authors’ data. b Based on: Castlebury & Carris (1999); Cunfer & Castlebury (1999); Milbrath et al. (1998); Castlebury (1998). c As T. barclayana: Castlebury & Carris (1999); CMI Description no. 75 (1965); Durán (1987); Durán & Fischer (1961). Or as T. horrida: Agarwal 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 Fig. 5). 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. It should be noted that teliospores can have a period of dormancy which can effect germination (Carris et al., 2006) and it should not be assumed that teliospores, which do not germinate, are not viable. 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 (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) (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), Carris et al. (2006), Table 2,Fig. 3, Appendix 311 The mounting medium, and heating or warming treatments, can affect teliospore size (Khanna & Payak, 1968; Agarwal et al., 1990; 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. . Figure 3Open in figure viewerPowerPoint 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). (3-5are at the same scale; 10 mm = 17 µm.) Other tuberculate-spored Tilletia species may be confused with T. indica (Durán & Fischer, 1961; Durán, 1987). In particularly, the morphologically and genetically similar fungus T. walkeri (ryegrass bunt), and also T. horrida (rice smut), are known contaminants of wheat seed or grain (Smith et al., 1996; Castlebury & Carris, 1999; Cunfer & Castlebury, 1999). 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 222 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 (Castlebury, 1998; Pimentel, 1998). : 3-5; Appendix 3). More than ten teliospores from a sample are needed to allow separation of species. Figure 4Open in figure viewerPowerPoint Teliospores of Tilletia walkeri (ryegrass bunt) showing surface ornamentation patterns: spines coarsely arranged and forming wide, incompletely cerebriform to coralloid ridges or thick clumps. (3-5are at the same scale; 10 mm = 17 µm.) Figure 5Open in figure viewerPowerPoint Teliospores of Tilletia horrida (rice smut) showing surface ornamentation patterns: polygonal scales or, occasionally, with cerebriform ridges. (3-5are at the same scale; 10 mm = 17 µm.) Principally T. horrida teliospores are 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 (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 (Fig. 6). Figure 6Open in figure viewerPowerPoint 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. 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 (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 (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 (Durán & Fischer, 1961; Pimentel et al., 1998). 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. Figure 7Open in figure viewerPowerPoint 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 h dark/light cycle. Note slower growth, and purple pigmentation after 14 days, for T. horrida colonies. If less than 10 teliospores are present then morphological characters are not considered totally reliable for confident discrimination. Furthering sample sieving should be performed to gain more than 10 teleospores or diagnosis should be confirmed using a molecular method as outlined below. Also when suspect teliospores are found the grains in both the washed subsample(s) and the larger submitted sample should be examined for Karnal bunt symptoms, but these are rarely observed. Molecular confirmation There are three main molecular methods available to confirm presumptive morphological diagnoses: 1 Restriction enzyme analysis of the ITS1 region (nrDNA Internally Transcribed Spacer Region 1) after PCR application using universal ITS primers (Appendix 5) 2 Conventional PCR assay using species-specific primers (Appendix 6) 3 PCR assay using species-specific primers and a fluorescent probe in a TaqMan system (Appendix 7). All these molecular confirmation methods require that teliospores are germinated and cultures produced from the resulting sporidia, which may take several weeks (Appendix 4). Detailed descriptions of these methods are enclosed in Appendix 5, 6 and 7. 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). Germination of teliospores for molecular confirmation may not always be possible, for example if grain is treated with NaOH as in the case of examining fungicide treated grain (Appendix 2) or teliospores are mounted in Shear's solution (Appendix 1). Increasing the number of sieved replicates may increase the number of teliospores recovered and hence the number of teliospore which can be germinated. 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 (Laroche et al., 1998; Pimentel 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 (Levy et al., 2001; Pimentel 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). Reference material Reference material can be provided on request from Dr. Kelvin Hughes at the Central Science Laboratory, York (GB). Report on the diagnosis Guidance on reporting and documentation is given in EPPO Standard PM 7/77 (1) Documentation and reporting on a diagnosis. Further information Further information on this organism can be obtained from: Pest and Disease Identification Team, Central Science Laboratory, Sand Hutton, York YO41 1LZ (GB). Footnotes 1 The mounting medium, and heating or warming treatments, can affect teliospore size (Khanna & Payak, 1968; Agarwal et al., 1990; 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. 2 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 (Castlebury, 1998; Pimentel, 1998). 3 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. Heurneman-van Brouwershaven, 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). 4 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. 5 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. 6 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. 7 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. 8 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. 9 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) approximately 1100 mv; free chlorine (5) – 10–15 (ppm). Equipment: Super Oxseed Labo, Advanced H2O Inc., Alameda, US. 10 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). N.B. Some teliospores can be killed if the total time in the bleach exceeds 2 min. 11 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 revision has been prepared by Kelvin Hughes. All methods except the molecular methods described in Appendices 5, 6 and 7 were ring-tested in different European laboratories33 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. Heurneman-van Brouwershaven, Plantenziektenkundige Dienst, Wageningen (NL); M. de Jesus Gomes, E. Diogo & M.R. Malheiros, Direcção-Geral de Protecção das Culturas, Lisboa (PT); V. 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