Simultaneous detection of Clavibacter michiganensis subsp. michiganensis, Pepino mosaic virus and Mexican papita viroid by non-radioactive molecular hybridization using a unique polyprobe
- 296 Downloads
Clavibacter michiganensis subsp. michiganensis, Pepino mosaic virus, and Mexican papita viroid are three economically important pathogens that infect tomato crops. In this work, a polyprobe (poly-3) was developed and evaluated for the simultaneous detection of these pathogens in tomato plants by non-isotopic molecular hybridization. The endpoint detection limit of the poly-3 with C. michiganensis subsp. michiganensis cell cultures was 106 CFU/ml. No differences were found in terms of the sensitivity and specificity when the individual riboprobes were compared to the poly-3 for the detection of the three pathogens. The analysis of 80 tomato field samples by the poly-3 and RT-PCR techniques rendered the same number of positive samples for each pathogen. As far as we know this is the first time that three pathogens with very different life cycle styles (bacteria, virus and viroid) are simultaneously detected in a single assay. The possibility of using this poly-3 technology for the routine diagnosis of field tomato samples is discussed.
KeywordsTomato Clavibacter michiganensis subsp. michiganensis Pepino mosaic virus Mexican papita viroid Polyriboprobe Non-isotopic molecular hybridization
E.J M-Z. was recipient of a Pre-doctoral fellowship from the Consejo Nacional de Ciencia y Tecnología of Mexico. F.A. was recipient of a contract Ramón y Cajal (RYC-2010-06169) Program of the Ministerio de Educación y Ciencia of Spain. We thank L. Corachan for her excellent technical assistance. This work was supported by Grants BIO2014-54862-R from the Spanish Granting Agency DGICYT, the Prometeo Program GV2011/003 from the Generalitat Valenciana and PAID-06-10-1496 from the Universitat Politecnica de Valencia (Spain).
- Anonymous. 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. Office for Official Publications of the European Communities. Consolidated Text, pages 1–159. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2000:169:0001:0112
- Astruc, N., Marcos, J. F., Macquaire, G., Candresse, T., & Pallás, V. (1996). Studies on the diagnosis of hop stunt viroid in fruit trees: identification of new hosts and application of a nucleic acid extraction procedure based on non-organic solvents. European Journal of Plant Pathology, 102, 837–846.CrossRefGoogle Scholar
- Chalupowicz, L., Cohen-Kandli, M., Dror, O., Eichenlaub, R., Gartemann, K.-H., Sessa, G., Barash, I., & Manulis-Sasson, S. (2010). Sequential expression of bacterial virulence and plant defense genes during infection of tomato with Clavibacter michiganensis subsp. michiganensis. Phytopathology, 100, 252–261.CrossRefPubMedGoogle Scholar
- Jorda, C., Pérez, A. L., Martıínez-Culebras, P., & Lacasa, A. (2001). First report of Pepino mosaic virus on natural hosts. Plant Disease, 85, 1292.Google Scholar
- Ling, K. & Carpenter, L. (2005). Pepino mosaic virus, an emerging disease in tomato greenhouse production worldwide; is seed responsible? Proc 1 sr IC on tomato diseases. In M.T. Momal, P. Ji and J.B. Jones (Eds.), Acta Horticultura 695.Google Scholar
- Özdemir, Z. (2009). Development of a multiplex PCR assay for the simultaneous detection of Clavibacter michiganensis subsp. michiganensis, Pseudomonas syringae pv. tomato and Xanthomonas axonopodis pv. Vesicatoria using pure cultures. Jouranl of Plant Pathology, 91, 495–497.Google Scholar
- Pallas, V., Sanchez-Navarro, J. A., Varga, F., Aparicio, F., & James, D. (2009). Multiplex Polymerase Chain Reaction (PCR) and Real-time Multiplex PCR for the simultaneous detection of plant viruses. Methods in Molecular Biology, 508, 193–208.Google Scholar
- Saade, M., Aparicio, F., Sanchez-Navarro, J. A., Herranz, M. C., Myrta, A., Di-Terlizzi, B., & Pallás, V. (2000). Simultaneous detection of the three ilarviruses affecting stone fruit trees by nonisotopic molecular hybridisation and multiplex reverse-transcription polymerase chain reaction. Phytopathology, 90, 1330–1336.CrossRefPubMedGoogle Scholar
- Thompson, J. R., Wetzel, S., Klerks, M. M., Vaskova, D., Schoen, C. D., Spak, J., et al. (2003). Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control. Journal of Virological Methods, 111, 85–93.CrossRefPubMedGoogle Scholar
- Verhoeven, J. ThJ., Roenhorst, J. W., & Owens, R. A. (2011). Mexican papita viroid and Tomato planta macho viroid belong to a sing le species in the genus Pospiviroid. Archives of Virology, 156, 1433–1437.Google Scholar
- Zhao, W. J., Chen, H. Y., Zhu, S. F., Xia, M. X., & Tan, T. W. (2007). One step detection of Clavibacter michiganensis subsp. michiganensis in symptomless tomato seeds using a TaqMan probe. Jouranl of Plant Pathology, 89, 349–351.Google Scholar