Abstract
A quantitative PCR assay based on the competitive PCR technique was compared to the classical soil dilution (SD) method for its ability to estimateV. dahliae propagules directly in soils collected from fields under potato production. A strong correlation (r = 0.97) was observed betweenV. dahliae propagules estimated using the quantitative PCR assay and those using the SD method. Coamplification ofV. dahliae DNA with competitor DNA provided accurate quantification in the range of 102 to 107 spores and 1 to 100 microsclerotia/g of soil. The number ofV. dahliae propagules detected in PEI soils ranged from 4.9 to 15.6 and 0.06 to 0.5 microsclerotia/g of soil for PCR assay and SD method, respectively. The strong correlation between PCR assay and SD method and the non significant differences between replications of PCR estimates ofV. dahliae propagules in soils (P< 0.05) show that the PCR assay is reliable and reproducible, and comparable to the SD method. This method is fast, does not depend on the subjectiveness of the traditional plating method, and offers an improvement in speed and precision over currently used methods. In addition, it can be extended to estimateV. dahliae propagules in other pathosystems and finds immediate and practical use in epidemiological studies to determine the effects of various crop management strategies on the dynamics and level of fungal propagules in the soil in order to establish threshold levels for assessing disease risks and develop disease prediction systems.
Resumen
Se comparó un ensayo cuantitativo de PCR basado en la técnica competitiva de PCR con el método clásico de dilucón del suelo (SD), por su capacidad de estimar propágulos de V. dahliae directamente en suelos colectados de otros campos de producción de papa. Se observó una fuerte correlación (r = 0.97) entre los propágulos de V. dahlia estimados usando el análisis cuantitativo de PCR con los usados en el método de dilución (DS). La co-amplificación del ADN de V. dahliae con el ADN competidor proporcionó cuantificación exacta en el rango de 102 a 107 esporas y de 1 a 100 microesclerotias por gramo de suelo. El número de propágulos de V. dahlie dectados en suelos PEI fluctuaron entre 4.9 a 15.6 y entre 0.06 a 0.5 microesclerotia/g de suelo por análisis de PCR y método de DS, respectivamente. La sólida correlación entre el análisis de PCR estimadas de los propágulos de V. dahlie en suelos (P<0.05), muestra que el análisis de PCR es confiable y reproducible y comparable con el método DS. Este método es rápido, no depende de las subjectividades de los métodos tradicionales de siembra y ofrece una mejoría en cuanto a velocidad y precisión respecto de otros métodos actualmente en uso. Además, puede expandirse par estimar los propágulos de V. dahlie en otros patosistemas y encontrar un uso inmediato y práctico en estudios epidemioloógicos par determinar los efectos de diversas estrategias de manejo de cultivos en la dinámica y nivel de propágulos de hongos en el suelo, con el fin de establecer niveles de inicio para evaluar los riesgos de la enfermedad y desarrollar sistemas de predicción de la enfermedad.
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Mahuku, G.S., Platt, H.W. QuantifyingVerticillium dahliae in soils collected from potato fields using a competitive PCR assay. Am. J. Pot Res 79, 107–117 (2002). https://doi.org/10.1007/BF02881519
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DOI: https://doi.org/10.1007/BF02881519