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

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.