Abstract
Isolation of DNA from obligate biotrophic soil-borne plant pathogens is challenging. This is because of their strict requirement of living plant tissue for their growth and propagation. A soil habitat further imposes risk of contamination from other microorganisms living in close vicinity of the plant roots. Here we present a protocol on how to prepare DNA suitable for advanced molecular analysis on the soil-borne pathogen Plasmodiophora brassicae, a peculiar unicellular plant pathogenic organism, causing disease on Crucifers. First, it is important to grow Brassica or Arabidopsis plants in infested soils below a temperature of 25 °C under moist conditions to promote root gall formation. Root galls should be harvested ahead of initiation of the decomposing process, no later than four or nine weeks post inoculation of Arabidopsis or Brassica plants, respectively. Resting spores with reduced numbers of soil organisms are achieved by gradient centrifugations of homogenized gall tissues. Treatments with 70% alcohol and a suit of different antibiotics promote P. brassicae purity. A CTAB-based procedure allows isolation of high quality DNA suitable for massive parallel sequencing analysis.
Keywords: Arabidopsis, Brassica, Clubroot, DNA, Plasmodiophora brassicae, Resting spores, Rhizaria
Background
Plasmodiophora brassicae is a soil-borne plant pathogen causing root galls (clubs) in the Brassicaceae family including Arabidopsis. The clubroot disease has a major impact on oilseed rape (canola) and cabbage cultivation worldwide. P. brassicae is an obligate biotroph (require a host for growth) assigned to the supergroup Rhizaria, one of the least studied organism groups of eukaryotes (Sierra et al., 2016; Sibbald and Archibald, 2017). Phylogenetically, P. brassicae belongs to a plant pathogenic group of protists in Phytomyxea (Neuhauser et al., 2011 and 2014; Adl et al., 2012). Few genomes of related species are available, a circumstance which has considerably delayed the molecular analysis and genome comparisons. P. brassicae forms hardy resting spores in the clubs, spores that have the capacity to remain dormant for decades in the soil, ready for new rounds of root infections if a host plant grow nearby. Here we describe how to generate diseased plants, isolate resting spores from root galls followed by extraction of large amounts of DNA. This protocol is a further improvement and clarification of the procedures described in Schwelm et al. (2015). The outlined work is substantial but yields high-quality DNA suitable for long-read massive parallel sequencing.
Materials and Reagents
Equipment
Procedure
Notes
Root infection varies much due to the environmental conditions and experiments should first be run to ensure proper club formation. The yield of resting spores and consequently of P. brassicae DNA vary depending on the club size and stage of maturation.
Recipes
Acknowledgments
We thank the plant growth facility staff at the Faculty of Natural Resources and Agricultural Sciences, Swedish University of Agricultural Sciences (SLU) for close attention of the plants. We also thank Dr. Arne Schwelm that started the extensive work of P. brassicae DNA preparation 2009. The authors declare that they have no conflicts of interest or competing interests. The work was funded by the Swedish Research Council (Formas) and SLU.
References
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