A rapid method for direct detection of Polymyxa DNA in soil

Abstract

Polymyxa spp. are vectors for a number of economically important soilborne plant viruses. The development of a technique to detect virus and vectors directly in soil would be useful for epidemiological studies and assessment of disease risk prior to planting. A rapid method was developed to extract and quantify Polymyxa spp. DNA from soils. DNA was extracted from three soils infested with Polymyxa betae and three infested with P. graminis using an EDTA lysis buffer in combination with a MagneSil™ DNA extraction kit and Kingfisher™ magnetic particle processor. Primers and probes designed to correspond to sequences within the internal transcribed spacer region 2 (ITS2) of ribosomal DNA enabled recovery and amplification of P. betae and P. graminis DNA using real-time PCR and TaqMan chemistry. For the P. graminis-infested soils, the purity of DNA obtained was sufficient to allow Polymyxa DNA to be amplified without dilution to remove inhibitors, but with P. betae-infested soils, amplification was only achieved if the DNA was diluted 1:10. Using TaqMan PCR, a standard curve was constructed from uninfested soil spiked with known numbers of P. betae cystosori; the quantity of P. betae inoculum from naturally infested soil was then extrapolated from the curve. This technique offers a sensitive method of extracting, detecting and quantifying Polymyxa spp. DNA in soil.

Management of plant health risks associated with processing of plant-based wastes: A review

“…in plant tissue is common e.g. Mutasa-Gottingens et al (1993, 1995, 2000, Ward et al (2004). However, this methodology is prone to false positive results due to the detection of small amounts of DNA in dead target material.…”

“…However, this methodology is prone to false positive results due to the detection of small amounts of DNA in dead target material. A rapid method for the detection of Polymyxa DNA directly from soil reported by Ward et al (2004) was capable of detecting as few as 2.78 × 10 3 cysts of P. betae g -1 soil, but was unable to distinguish viable from nonviable cystosori. Kingsnorth et al (2003) developed a recombinant antibody ELISA assay for the P. betae glutathione-S-transferase, which is expressed during infection.…”

“…Phytophthora kernoviae sporangia oospores similar to P. ramorum no data Schena et al (2006) Polymyxa spp cystosori in roots or soil 100-150 cystosori detection of spores from soil using Real Time PCR 1.39 × 10 3 cystosori g -1 soil Ward et al (2004) bait tests followed by glutathione-S-transferase ELISA or microscopy no data √ Kingsnorth et al (2003) MPN method bait test method 50-100 cystosori √ Tuitert (1990) 58 Roenhorst et al (2005) …”

On-Site DNA Extraction and Real-Time PCR for Detection of Phytophthora ramorum in the Field

“…A nested real-time PCR assay for P. ramorum using SYBR green (a double-stranded DNAbinding dye [as DNA is amplified, more dye is bound and thus fluoresces]) has been described (14), and an assay using molecular beacons (probes containing reporter and quencher dyes which hybridize to the amplified product, resulting in increased fluorescence) is in development (5). Real-time PCR methods based on TaqMan chemistry (amplification-dependent cleavage of probes incorporating reporter and quencher dyes, resulting in increased fluorescence) have the particular advantage of requiring no postamplification steps and therefore involve a reduced risk of cross-contamination, and they have been described for a wide range of plant pathogens (26,30,32,33,34). A single-round TaqMan PCR assay for the detection of P. ramorum has recently been developed which compares extremely favorably with morphological methods of identification (K. J. D. Hughes, R. L. Griffin, J.…”

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

“…In particular, real-time PCR methods have the advantages of speed, accuracy, and sensitivity over other methods (7,28,30,43) and can be closedtube systems involving no postamplification steps. TaqMan (11,16) is the most widely used real-time PCR system, and assays using this detection chemistry have been described for a range of plant pathogens (3,4,14,39,38,40), including assays for the detection of Phytophthora ramorum both in the laboratory (8,12,34) and in the field (33). A single round of real-time PCR has been found to be sufficiently sensitive to achieve the detection of 10 to 100 fg P. ramorum DNA in plant material (8,33).…”