1. Quantification of Plasmodiophora brassicae Resting Spores in Soils Using Droplet Digital PCR (ddPCR)
- Author
-
Rui Wen, Bruce D. Gossen, Nazmoon Tonu, Tim J. Dumonceaux, Gary Peng, Mingguang Chu, Fengqun Yu, and Jillian Lee
- Subjects
0106 biological sciences ,0301 basic medicine ,Soil test ,fungi ,food and beverages ,Soil classification ,Plant Science ,Biology ,medicine.disease ,01 natural sciences ,Potting soil ,Spore ,Clubroot ,03 medical and health sciences ,Horticulture ,030104 developmental biology ,Soil water ,medicine ,Bioassay ,Digital polymerase chain reaction ,Agronomy and Crop Science ,010606 plant biology & botany - Abstract
Plasmodiophora brassicae, an obligate soilborne pathogen that causes clubroot on Brassica crops, is spreading rapidly in western Canada, threatening canola production in the region. Bioassays and molecular assays have been used to estimate the concentration of P. brassicae resting spores in soil, which can affect clubroot incidence and severity on crops. Droplet digital PCR (ddPCR) is a promising new approach for quantification of pathogen inoculum owing to its low sensitivity to inhibitors and consistency at low target concentrations. The objective of this study was to assess ddPCR against existing quantitative PCR (qPCR) for potential advantage and/or improvement in quantifying P. brassicae resting spores in soil. The new protocol enumerated resting spores accurately in spiked potting mix or soil samples ranging from 102 to 107 spores per gram. At a spore concentration ≥107 spores per gram, however, ddPCR became less accurate, with a tendency of overestimation. The protocol was validated by quantifying the resting spores in spiked brown, dark brown, and black soils using both ddPCR and qPCR simultaneously. These soil types are found commonly on the Canadian Prairies, and they vary in texture, pH, and organic content. ddPCR showed similar results among the different soil types, whereas qPCR often displayed lower counts for the same spore concentration, with the amplification of DNA inhibited completely in black soil samples. The inhibition can be removed by a 10-fold dilution of DNA samples. The results show that ddPCR can be a more versatile tool than qPCR for detection and quantification of P. brassicae resting spores in soil samples.
- Published
- 2020
- Full Text
- View/download PDF