Quantitative detection of Monosporascus cannonballus in infected melon roots using real-time PCR
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A method was developed for the specific detection, identification and quantification of Monosporascus cannonballus in infected melon roots based on real-time PCR (SYBR® Green chemistry) targeting the ITS1 region of the rDNA conserved between different strains of the pathogen. The specificity of the reaction was assessed using a number of fungi taxonomically and ecologically related to M. cannonballus. The method was highly sensitive and M. cannonballus was first detected in the roots of a susceptible Piel de Sapo cultivar 2 days after inoculation, before symptom appearance. Although conventional PCR methods could also provide such a specific and sensitive detection, real-time PCR was also able to produce reliable quantitative data over a range of 4 orders of magnitude (from 5 ng to 0.3 pg). The method allowed the quantitative monitoring of fungal growth from the very first stages of infection, and was successfully employed in the early screening of resistance. The assessment of disease progress and severity obtained with real-time PCR was more accurate than that obtained with the visual scoring of root lesions or root biomass losses. Therefore, there exists a great potential for its implementation in those steps of breeding programmes where high accuracy is required.
KeywordsAscomycetes Cucumis melo Disease assessment ITS-regions Root rot/vine decline SYBR® Green
The authors would like to thank Eva Ma Martínez (COMAV) for her technical assistance with the infected material, and to Dr. Jose Miguel Blanca and Dr. Alicia Sifres (COMAV) for their help with real-time PCR. This work was supported by MCT AGL2003-04817 and GEN2003-20237-C06-03. We would also like to thank the plant pathology group at the UPV, Dr. B.D. Bruton, and Dr M. Stanghellini for providing some fungal isolates.
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