In‐field distribution of Plasmodiophora brassicae measured using quantitative real‐time PCR

Abstract

A protocol using real-time polymerase chain reaction (PCR) for the direct detection and quantification of Plasmodiophora brassicae in soil samples was developed and used on naturally and artificially infested soil samples containing different concentrations of P. brassicae. Species-specific primers and a TaqMan fluorogenic probe were designed to amplify a small region of P. brassicae ribosomal DNA. Total genomic DNA was extracted and purified from soil samples using commercial kits. The amount of pathogen DNA was quantified using a standard curve generated by including reactions containing different amounts of a plasmid carrying the P. brassicae target sequence. The PCR assay was optimized to give high amplification efficiency and three to four copies of the target DNA sequence were detected. Regression analysis showed that the standard curve was linear over at least six orders of magnitude (R 2 > 0AE99) and that the amplification efficiency was >92%. The detection limit in soil samples corresponded to 500 resting spores g )1 soil. The intersample reproducibility was similar to, or higher than, that of assays for other pathogens quantified in soil samples. Bait plants were used to validate the real-time PCR assay. The protocol developed was used to investigate the spatial distribution of P. brassicae DNA in different fields and a significant difference was found between in-field sampling points. The reproducibility of soil sampling was evaluated and showed no significant differences for samples with low levels of inoculum, whereas at higher levels differences occurred. Indicator kriging was used for mapping the probability of detecting P. brassicae within a 2-ha area of a field. A threshold level of 5 fg plasmid DNA g )1 soil, corresponding to approximately 3 · 10 3 P. brassicae resting spores g )1 soil, is suggested for growing resistant cultivars. The results provide a robust and reliable technique for predicting the risk of disease development and for assessing the distribution of disease within fields.

Pathotypes of Plasmodiophora brassicae causing damage to oilseed rape in the Czech Republic and Poland

“…This likely reflects the fact that the qPCR method is more robust and has greater sensitivity. Similar results were reported by Wallenhammar et al (2012), where the detection limit in soil samples corresponded to 500 resting spores/g soil.…”

Spatial distribution of cabbage root maggot (Delia radicum) and clubroot (Plasmodiophora brassicae) in winter oilseed rape crops in the Czech Republic

“…They also consider the use of bait plants as the most reliable diagnostic method for assessing soils for the presence of P. brassicae, despite the availability of more modern techniques (Faggian & Strelkov 2009). Contrary to Wallenhammar et al (2012) we did not assess the abundance of P. brassicae spores in soils, but through the expression of symptoms on the plants (susceptible varieties) always sampled from the all regions (rectangular grids) of the compared fields and through the usage of SADIE (Perry 1995(Perry , 1996, our results also indicate the patchiness of P. brassicae distribution within fields -but only there where lower levels of disease incidence in crops were recorded. Říčařová et al (2016) also documented a tendency of P. brassicae spores (inoculum) and clubroot symptomatic plants to aggregate to one or more focuses in fields.…”

“…In crops with higher levels of clubroot incidence, the symptomatic plants show a tendency for random (Nový Malín 2014, Bludov 2014 or almost uniform (Libina 2012, practically the whole crop infested at the same level) distribution in crops. Wallenhammar et al (2012) studied the distribution of P. brassicae spores (inoculums) in arable soil in Sweden. They demonstrate the patchiness of inoculum distribution even within a small area and emphasise the importance of including an adequate number of subsamples when sampling.…”

“…Both the organisms can aggregate into one or more foci in fields. At such places they show significantly higher abundances (Jones et al 1993;Wallenhammar et al 2012;Seidenglanz et al 2013;Říčařová et al 2016). …”

“…The damage induced by them and their importance for growers has substantially increased in the Czech Republic recently (Říčařová et al 2016). Clubroot in brassica crops, caused by the obligate endoparasite P. brassicae, is recognised as a serious soilborne disease (Wallenhammar et al 2012;Říčařová et al 2016), usually associated with appreciable yield losses (Wallenhammar 1998;Wallenhammar et al 2012;Říčařová et al 2016). A well known symptom of the damage induced by P. brassicae visible on winter oilseed rape plants is their abnormally swollen roots (tumours occur on the main root and even adjacent roots), which cannot then properly supply the above ground plant parts with water and nutrients.…”