Several studies have examined the viability of resting spores by fluorescent dyes, such as the cellulose and chitin binding fluorochrome calcofluor white and the nucleic acid stain ethidium bromide ( Takahashi and Yamaguchi, 1988). However, such methods have not been systematically explored and evaluated and thus are lacking so far. Accurate methods for assessing resting spore viability and germination are therefore essential in any studies aiming at the development of effective measures of disease control. The poor knowledge on the nature and function of such regulatory factors–to a significant extend–is due to a lack of accurate and reliable methods to study spore activities. Thus, the regulation of resting spore germination is a crucial factor for the completion of the pathogen life cycle through infection and disease induction. Under favorable conditions, resting spores are induced to germinate and release primary biflagellate zoospores, which are required for infection in the field. This is the only stage in which the pathogen can survive in the absence of host plants. brassicae can survive for a long period in the soil as resting spores. brassicae, survival in the soil as resting spores, root hair infection, and cortical invasion ( Figure 1). There are three stages in the life cycle of P. brassicae in the soil prior to infection. 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. Most clubroot resistance sources are race-specific and have not been durable, since virulent clubroot isolates (e.g., pathotype 5×) have been found which overcome the race-specific clubroot resistance ( Diederichsen et al., 2009 Rahman et al., 2014). The efficacy of biocontrol treatments in the field is limited ( Tremblay et al., 2005 Zhou et al., 2014 Yu et al., 2015). However, there are no approved pesticides against clubroot available in Europe. 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). brassicae resting spores also results in a reduction in land capital value and revenue when fields are infected. This disease leads to considerable losses in yield and quality. Infected plants become stunted and wilt, while the roots are distorted and swollen, compromised in the uptake of water and nutrients. Plasmodiophora brassicae is an obligate phytopathogen that causes clubroot disease on cruciferous crops. brassicae resting spores causing clubroot disease in Brassicaceae hosts and therefore are fundamental to develop novel strategies of control. Such methods are required to study factors in the soil regulating survival, dormancy and germination of P. This study evaluates and confirms the suitability of various microscopic and molecular genetic methods for the determination of viability and germination of P. Alternatively, dual staining with CFW-Nile red was successfully used to assess the germination rate of resting spores with a lethal pre-treatment. The latter method also allowed recording individual germination states of spores. 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. 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. CFW-PI was capable of reliably determining the viability within 10 min, while Evans blue required overnight incubation to obtain accurate results. Dual staining with calcofluor white-propidium iodide (CFW-PI) or single stain with Evans blue showed reliable accuracy in estimating viability. In this study, we evaluated several methods for the assessment of viability and germination rate of P. The accurate assessment of viability and germination rate is the foundation to evaluate the effect of control methods. The viability and germination rate of resting spores are crucial factors of the inoculum potential in the field. brassicae can survive in the soil for a long period without hosts or external stimulants.
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