Abstract
Clubroot caused by the obligate biotrophic parasite Plasmodiophora brassicae is a destructive soil borne disease of cruciferous crops. Resting spores of P. brassicae can survive in the soil for a long period without hosts or external stimulants. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. In this study, we evaluated several methods for the assessment of viability and germination rate of P. brassicae resting spores. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Due to DNA degradation of heat treatments, acetone was selected to evaluate the efficiency of propidium monoazide (PMA)–quantitative PCR (qPCR) used for the quantification of DNA from viable cells. The staining with 4,6-Diamidine-2-phenylindole dihydrochloride (DAPI) and the use of differential interference contrast microscopy were suitable for the determination of resting spore germination rates. The latter method also allowed recording individual germination states of spores. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. brassicae resting spores. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control.
Highlights
Plasmodiophora brassicae is an obligate phytopathogen that causes clubroot disease on cruciferous crops
Both staining methods of Evans blue and calcofluor white-propidium iodide (CFW-PI) showed good performance to distinguish viable from non-viable spores
To provide powerful tools for life cycle studies of P. brassicae, several methods were evaluated for their ability to assess resting spore viability, including fluorescein diacetate, trypan blue, methylene blue, acridine orange, Evans blue and CFW-PI as well as propidium monoazide (PMA) quantitative PCR (qPCR)
Summary
Plasmodiophora brassicae is an obligate phytopathogen that causes clubroot disease on cruciferous crops. Infected plants become stunted and wilt, while the roots are distorted and swollen, compromised in the uptake of water and nutrients. This disease leads to considerable losses in yield and quality. Due to the severe damage caused by P. brassicae and its widespread geographic distribution, a variety of control methods have been developed, such as soil liming (Tremblay et al, 2005), crop rotation (Friberg et al, 2006; Hwang et al, 2015; Peng et al, 2015), chemical treatments (Suzuki et al, 1995; Tanaka et al, 1999; Mitani et al, 2003), cultivation of resistant cultivars (Kuginuki et al, 1999; Piao et al, 2004; Hirai, 2006), and biocontrol (Lahlali et al, 2013; Lahlali and Peng, 2014). The development of novel, more effective strategies of control is hindered by a lack of accurate methods to study crucial stages in the life cycle of P. brassicae in the soil prior to infection
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