Simultaneous detection of Clavibacter michiganensis subsp. michiganensis, Pepino mosaic virus and Mexican papita viroid by non-radioactive molecular hybridization using a unique polyprobe
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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).
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