Improved extraction of Rhizoctonia and Pythium DNA from wheat roots and soil samples using pressure cycling technology

Abstract

Soilborne pathogens are important biotic factors in yield reduction in the dryland cereal production region of the Pacific Northwest. Rhizoctonia solani AG-8, Rhizoctonia oryzae, and Pythium spp. are causal agents of root rot, bare patch, and damping-off of wheat (Triticum aestivum) and barley (Hordeum vulgare). Although these pathogens can be rapidly and specifically quantified using quantitative real-time PCR, the extraction of Rhizoctonia DNA from agricultural samples is often inconsistent, especially at low pathogen population densities. Using a novel extraction system that uses pressure cycling technology (PCT), we improved the extraction of R. solani AG-8 DNA up to 16-fold and of P. abappressorium DNA up to 2-fold from three types of agricultural soils compared with a bead beating extraction method. PCT also yielded quantifiable amounts of R. solani AG-8 and R. oryzae DNA from lyophilized wheat roots that were otherwise recalcitrant to homogenization. Furthermore, the extractions were so consistent that pathogen quantification generally could be derived from two rather than three or four replicated extracts. Because PCT is performed in a closed system and minimizes sample shearing and heating, it confers a substantial advantage over conventional extraction systems. Here, we report for the first time the application of PCT in a laboratory setting for the improved extraction and quantification of three types of soilborne pathogens in soil samples. The effectiveness of PCT for three soils suggests that it will be beneficial for other hard-to-extract pathogen samples.

Keywords:

• Barocycler^TM
• necrotrophic pathogen
• PULSE tube^TM
• real-time PCR
• root disease
• soilborne pathogens