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Detection and quantification ofSpongospora subterranea f. sp.subterranea by PCR in host tissue and naturally infested soils

  • Xinshun Qu
  • James A. Kavanagh
  • Damian Egan
  • Barbara J. Christ
Article

Abstract

A polymerase chain reaction (PCR) assay using primers SsF and SsR designed from the internal transcribed spacer (ITS) regions ofSpongospora subterranea f. sp.subterranea was developed for the specific identification and quantification ofS. subterranea. These primers amplified a 434 bp product from DNA ofS. subterranea spore balls, but not from DNA of healthy potato, common scab tuber, and taxonomically related plasmodiophorids. This PCR assay was successfully used for the detection ofS. subterranea in naturally infected symptomatic and asymptomatic potato tubers.Spongospora subterranea in other infected symptomless host plants was detected by PCR. The PCR assay was modified with improved soil DNA extraction methods to detectS. subterranea in soil. The assay was sensitive, and one spore ball per gram of soil could be detected. Following the design of a heterologous competitor DNA template from the sequence of λDNA, a competitive PCR assay for the quantification ofS. subterranea in soil was developed and provided accurate quantification in the range of 1 to 104 spore balls per 0.25 g of soil. In a preliminary survey of naturally infested field soil samples, spore ball concentrations were estimated to vary from ca 0 to 3600 spore balls per 0.25-g soil sample by this competitive PCR assay. The spore ball levels were compared with the powdery scab disease incidence of potatoes in these fields, and a correlationship between spore ball levels and subsequent disease incidence was found. The PCR assays developed in this investigation can be routinely used to detect and quantifyS. subterranea in diseased plant tissue, asymptomatic plant tissue, and infested soil.

Additional key words

Solanum tuberosum 

Resumen

La prueba de reacción en cadena de la polimerasa (PCR) utilizando los “primers” SsF y SsR, diseñados a partir de las regiones transmitidas internamente del espaciador (ITS) deS. subterránea f. sp.subterranea, ha sido desarrollada para la identificación específica y cuantificación deSpongospora subterranea. Estos “primers” amplificaron el producto 434 bp del DNA de las masas de esporas deS. subterranea pero no del DNA de papa sana, o de tubérculos con sarna común y plasmodiophoridos taxonómicamente relacionados. Esta prueba de PCR ha sido utilizada exitosamente para la detección deS. subterranea en tubérculos naturalmente infectados, sintomáticos y asintomáticos. También se ha detectado a través del PCR,S. subterranea en otros huéspedes infectados que no presentaban síntomas. La prueba PCR ha sido modificada con métodos mejorados de extracción de DNA del suelo para detectarS. subterranea. La prueba es tan sensible que pude detectar una espora por gramo de suelo. Después del diseño de una muestra competitiva heteróloga de DNA de la secuencia de λDNA se desarrolló una prueba competitiva de PCR para la cuantificaciónS. subterranea en el suelo, la misma que proporcionó una cuantificación segura en el rango de 1 a 104 masas de esporas por 0.25 gramos de suelo. En un estudio preliminar de muestras de suelo infestado naturalmente, se estimó que la concentración varió de c. 0 a 3600 masas de esporas por 0.25 gramos de muestra de suelo por medio de esta prueba de PCR. El nivel de masas de esporas se comparó con la incidencia de sarna polvorienta en estos campos de papa y se encontró una correlación entre los niveles de masas de esporas e incidencia de la enfermedad en una siguiente campaña. Las pruebas de PCR desarrolladas en esta investigación pueden ser rutinariamente utilizadas para detectar y cuantificarS. subterranea en el tejido de plantas enfermas, tejido de plantas asintomáticas y suelo infestado.

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Copyright information

© Springer 2006

Authors and Affiliations

  • Xinshun Qu
    • 1
  • James A. Kavanagh
    • 2
  • Damian Egan
    • 2
  • Barbara J. Christ
    • 1
  1. 1.Department of Plant PathologyThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.School of Biology and Environmental Science, Agricultural and Food Science CentreUniversity College DublinDublin 4Ireland

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