A one-tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat Mui-Keng Tan A,B,H , Aida Ghalayini A,B , Indu Sharma C , Jianping Yi D , Roger Shivas B,E , Michael Priest B,F and Dominie Wright B,G A Elizabeth Macarthur Agricultural Institute (EMAI), NSW Department of Primary Industries, PMB 8, Camden, NSW 2570, Australia. B CRC for National Plant Biosecurity, 2/4 Phipps Close, Deakin, ACT 2600, Australia. C Department of Plant Breeding, Genetics and Biotechnology, Punjab Agricultural University, Ludhiana 141004, Punjab, India. D Shanghai Entry-Exit Inspection and Quarantine Bureau, 1208 Minsheng Road, Pudong New Area, Shanghai 200135, China. E Plant Pathology Herbarium, Department of Primary Industries and Fisheries, Indooroopilly, Qld 4068, Australia. F Orange Agricultural Institute, NSW Department of Primary Industries, Orange, NSW 2800, Australia. G Department of Agriculture and Food, Locked Bag 4, Bentley Delivery Centre, WA 6983, Australia. H Corresponding author. Email: mui-keng.tan@dpi.nsw.gov.au Abstract. A molecular assay with enhanced specificity and sensitivity has been developed to assist in the surveillance of Karnal bunt, a quarantineable disease with a significant impact on international trade. The protocol involves the release of DNA from spores, PCR amplification to enrich Tilletia-specific templates from released DNA and a five-plex, real-time PCR assay to detect, identify and distinguish T. indica and other Tilletia species (T. walkeri, T. ehrhartae, T. horrida and a group comprising T. caries, T. laevis, T. contraversa, T. bromi and T. fusca) in wheat grains. This fluorescent molecular tool has a detection sensitivity of one spore and thus bypasses the germination step, which in the current protocol is required for confirmation when only a few spores have been found in grain samples. The assay contains five dual-labelled, species-specific probes and associated species-specific primer pairs in a PCR mix in one tube. The different amplification products are detected simultaneously by five different fluorescence spectra. This specific and sensitive assay with reduced labour and reagent requirements makes it an effective and economically sustainable tool to be used in a Karnal bunt surveillance program. This protocol will also be valuable for the identification of some contaminant Tilletia sp. in wheat grains. Introduction Karnal bunt of wheat is caused by the smut fungus Tilletia indica Mitra and was first found in Karnal, India (Mitra 1931, 1935). The pathogen causes only a slight reduction in yield but adversely affects wheat quality and marketability due to the production of trimethylamine, which gives the grain a fishy odour. The fungus is thus subjected to very strict quarantine regulations by countries that are not known to have the fungus, including Australia. Any possible incursion would cause severe disruption to Australia’s international wheat trade. The current diagnostic protocol (Inman et al. 2003; Wright et al. 2003) involves the tentative identification of the spores based on morphology followed by germination of the spores and a molecular protocol to confirm the identity. Microscopy and spore germination are very time consuming and labour intensive. Tilletia spp. reported as contaminants in Australian wheat grain included T. caries (DC.) Tul. & C. Tul., T. laevis J.G. Kühn, T. ehrhartae P.H.B. Talbot, and T. walkeri L.A. Castlebury & L.M. Carris (Pascoe et al. 2005). Conventional taxonomic separation based on colour, spore sizes and ornamentation is labour intensive and requires either considerable expertise or a significant quantity of ~50 spores of each species for a statistically significant determination (Inman et al. 2003). The fungus T. walkeri (Castlebury and Carris 1999) is morphologically similar to T. indica and infects ryegrass but does not infect wheat and so unequivocal differentiation from T. indica is critical from a quarantine perspective. The smut fungus T. ehrhartae (Pascoe et al. 2005) infects Ehrharta calycina (perennial veldt grass) in southern Australia and South Africa. Although T. ehrhartae ustilospores are smaller (<25 mm diam.) than T. indica, the resemblance of their surface ornamentations is a cause for possible misidentifications if only a very few spores are seen. The frequent presence of spores of both T. ehrhartae and T. walkeri in Australian wheat grain for export has significant implications for the testing of Australian export wheat using the existing protocols (Frederick et al. 2000; CSIRO PUBLISHING www.publish.csiro.au/journals/app Australasian Plant Pathology, 2009, 38, 101–109 Ó Australasian Plant Pathology Society 2009 10.1071/AP08088 0815-3191/09/020101