Quantification of potato common scab pathogens in soil by quantitative competitive PCR with fluorescent quenching‐based probes

Abstract

Common scab of potato tubers caused by pathogenic Streptomyces spp. is a cause of serious economic loss worldwide. For the rapid and accurate quantification of pathogenic Streptomyces spp. residing in soil, a new competitive real-time PCR method using fluorescent quenching-based probes (quantitative competitive quenching probe PCR: QCQP-PCR) was developed. The virulence gene of pathogenic Streptomyces spp., nec1 , was selected as the target for QCQP-PCR. A specific primer set to amplify the nec1 gene, and a fluorescently labelled probe that specifically hybridizes with the nec1 amplicon were designed. For QCQP-PCR, an internal standard DNA (IS DNA) that is identical to the nec1 amplicon but has a 4-base mismatch in the probe-hybridizing region, and a fluorescently labelled probe IS, which specifically hybridizes with IS DNA at the mutagenized region, were PCR-synthesized. The target nec1 gene was co-amplified with the known copy number of IS DNA by PCR using the same primer set in the presence of the specific probes. The PCR products were monitored in real-time by measuring the fluorescence intensity (quenching) of each probe. The initial amount of the nec1 gene was quantified based on the ratio of the PCR products of the same PCR cycle. The results revealed that QCQP-PCR could be used to precisely quantify the nec1 gene, even in the presence of PCR inhibitors in the soil samples examined. The lower limit of quantification was 20 copies per tube, which corresponded to 1500 copies per g dry soil. The quantification achieved by this method was completed within 5 h, i.e. the duration of the entire analysis. These results demonstrate the usefulness of the present method for monitoring pathogenic Streptomyces species in soil.

Comparison between Pathogenic <i>Streptomyces scabies</i> Isolates of Common Scab Disease

“…They were identified as Streptomyces scabies. These results are in agreement with those noted by several researchers who reported that S. scabies was responsible for common scab of potato (Lambert and Loria, 1989;Goyer and Beaulieu, 1997;Bencheikh and Setti, 2007;Manome et al, 2008).…”

Community Analysis of Root- and Tuber-Associated Bacteria in Field-Grown Potato Plants Harboring Different Resistance Levels against Common Scab

“…In order to achieve the successful biological control of plant diseases, including potato common scab, it is also important to understand the ecology and epidemiology of pathogens through the specific detection and quantification of pathogenic Streptomyces on potato tissue and in soil under field conditions. A TaqMan real-time PCR assay using virulence gene nec1 ( 5 ), SYBR Green quantitative real-time PCR assay using the txtAB operon ( 33 ), and quantitative competitive quenching probe PCR assay using the nec1 gene ( 26 ) have been used for the accurate detection and quantification of pathogenic Streptomyces in the potato. Although a good correlation has been reported between the presence of nec1 and pathogenicity, some nonpathogenic nec1 -positive strains and numerous pathogenic nec1 -negative strains have been described ( 33 ).…”

Diversity Analysis of Streptomycetes and Associated Phosphotranspherase Genes in Soil

“…The atpD sequence data suggest that the most common streptomycin producer from isolation work, S. griseus DSM 40236 and its close relatives, is found at a low density in soil.However none of the cloned atpD genes matched or were related to S. griseus DSM 40236. The molecular detection limit for plant pathogenic streptomycetes, , in soil has been calculated to be 10 3 gene targets per gram of soil for the txtAB gene [38] , 1.5×10 3 for the nec1 gene [39] and 10 2 for the 16 S gene [40] , suggesting that S. griseus soil populations may be lower than these values even after enrichment with chitin and starch. Other studies also support this finding as TCDNA studies of 13 streptomycetes from soil crusts in the Colorado Plateau [41] , 156 streptomycetes from prairie soil in the Cedar Creek Natural History Area [42] , and an isolate study including 3204 isolates from Singapore rainforests [43] failed to detect S. griseus , while a TCDNA study of temperate forest soils from Italy found one S. griseus sequence among 22 streptomycetes [44] , indicating that S. griseus is uncommon in soil.…”