Assessment of Factors Associated with Molecular Quantification of Stubby Root Nematode Paratrichodorus allius from Field Soil DNA
| Autor: | Danqiong Huang, Jonathan L. Whitworth, Guiping Yan, Neil C. Gudmestad |
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| Rok vydání: | 2019 |
| Předmět: | |
| Zdroj: | Plant Disease. 103:3265-3273 |
| ISSN: | 1943-7692 0191-2917 |
| DOI: | 10.1094/pdis-12-18-2240-re |
| Popis: | Factors relating to SYBR Green-based quantitative real-time PCR (qPCR) quantification of stubby root nematode Paratrichodorus allius using soil DNA were evaluated in this study. Soils used were loamy sand from potato fields in North Dakota and Idaho. Results showed that the largest nematode individuals (body length >720 µm) produced significant lower Cq values than the smallest individuals (4)(SO4)2 treatment during DNA preparation significantly reduced the inhibitors compared with post-treatment of soil DNA with polyvinylpolypyrrolidone column. The effect of PCR inhibitors on qPCR was suppressed by bovine serum albumin. Quantification results did not significantly change when increasing the number of DNA extractions from three to six per soil sample when soil grinding and grid sampling strategies were used. Two standard curves, generated from serial dilutions of plasmid DNA containing P. allius ITS1 rDNA and soil DNA containing known nematode numbers, produced similar correlations between Cq values and amount of targets. The targets in soil DNA quantified by qPCR using either standard curve correlated well with microscopic observations using both artificially and naturally infested field soils. This is the first study for assessing various factors that may affect qPCR quantification of stubby root nematodes. Results will be useful during the setup or optimization of qPCR-based quantification of plant-parasitic nematodes from soil DNA. |
| Databáze: | OpenAIRE |
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