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Publication datasheet
Title:
Comparison of molecular diagnostic methods for the identification of the quarantine pathogen Tilletia indica mitra
Authors:
Bragaloni, M.; Riccioni, L.
Year:
2010
Languages:
ENG, eng
Journal:
Petria
Kind of publication:
Cartaceo
Location:
Roma
Editor:
CRA-PAV
Abstract in Italian:
Abstract in English:
Tilletia indica Mitra is a seed-borne quarantine plant pathogen fungus agent of the Karnal bunt of wheat. The fungus, which originally spread from the Asian and later in the American Continent, is actually inserted in the A1 list of European and Mediterranean Plant Pathology Organisation (EPPO), since, the introduction in European Union, where the pathogen is absent up today, is considered of high risk (Directive 2000/29/CE). Methods for diagnosis have been developed by EPPO (OEPP/EPPO 1990, 1991, 2004, 2007)) in order to check the unwelcome introduction in the above countries of the pathogen. However, the morphological identification of the Tilletia indica has to be considered difficult for the potential co-presence of other Tilletia species especially as the content of teliospores, available in the seed samples under diagnosis, is poor. Since ryegrass seeds and wheat seeds are usually transferred by ship, the contamination of the T. walkeri on wheat seeds is a possible event. Moreover, the incapacity to well-distinguish the two species by morphological criteria for those samples which have a poor teliospore content could cause misidentification and/or false alarmism for the presence of T. indica. Different molecular methods were developed (Pimentel et al., 1998 Frederick et al., 2000), and introduced in the PM 7/29 protocol. However, the above methods need DNA extracted from mycelium, but the low percentage of germination because of the dormancy and the poor content of teliospores which can frequently result from the standardize 50 grams seed sample used for the “washing test” are factors which may negatively affect the species identification. A very recent and promising one-tube fluorescent assay by Real Time PCR was developed for multiplex DNA identification of Tilletia species by Mui-Keng Tan et al., (2009). The above method and some of the EPPO methods were tested on teliospores and/or DNA of T. indica and T. walkeri in order to compare the ability of the different protocols. The results in term of species identifications, advantages and limits of each protocol are showed and discussed. Emphasis and discussion is more focalised upon the use of Tan et al. protocol for the detection of the two species in seed samples with low-teliospore content by performing the analysis directly on a crushed single-spore as starting material without performing any DNA extraction procedure. Key words: Triticum spp., Lolium spp., Real Time PCR, Karnal or Partial Bunt of Wheat, Identification. Acknowledgements This study was carried out within the programme ARNADIA-ARON “Armonizzazione della diagnosi e valutazione del rischio di patogeni da quarantena e nocivi ai vegetali e ai prodotti vegetali”, financed by the Ministero delle Politiche Agricole Alimentari e Forestali, Italy. References Council Directive 2000/29/EC of 8 May 2000 on protective measures against the introduction into the Community of organisms harmful to plants or plant products and against their spread within the Community: Official Journal of the European Communities, 43, I. 169 1-112. OEPP/EPPO (1990) Specific quarantine requirements. EPPO Technical Documents No. 1008. OEPP/EPPO (1991) Quarantine procedure No. 37. Tilletia indica. Inspection and test methods for wheat seeds. Bulletin OEPP/EPPO Bulletin, 21, 265-266. OEPP/EPPO (2004) Diagnostic protocols for regulated pests, Protocoles de diagnostic pour les organismes réglementés - PM7/29 Tilletia indica. Bulletin OEPP/EPPO Bulletin, 34, 155 –157. OEPP/EPPO (2007) PM7/29(2) Tilletia indica. Bulletin OEPP/EPPO Bulletin, 37, 503–520. Frederick RD, Snyder KE, Tooley PW, Berthier-Schaad Y, Peterson GL, Bonde MR, Schaad NW & Knorr DA (2000) Identification and differentiation of Tilletia indica and T. walkeri using the polymerase chain reaction. Phytopathology, 90, 951–960. Pimentel G, Carris LM, Levy L & Meyer R (1998) Genetic variability among isolates of Tilletia barclayana, T. indica and allied species. Mycologia, 90, 1017–1027. Tan MK, Ghalayini A, Sharma I, Jianping Y, Shivas R, Priest M, Wright D. 2009. A one tube fluorescent assay for the quarantine detection and identification of Tilletia indica and other grass bunts in wheat. Australasian Plant Pathology, 38,101-109.

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