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Efficient quantification of Globodera pallida and G. rostochiensis (Tylenchida: Heteroderidae) in large amounts of soil using probe‑based real‑time PCR
Get PDFReal-time quantitative polymerase chain reaction (qPCR) was used to estimate the population densities of the potato cyst nematodes Globodera pallida Stone and Globodera rostochiensis (Wollenweber) Skarbilovich (Tylenchida: Heteroderidae). Since it is difficult to extract nematode DNA from large amounts of soil (≥ 100 g, enough for quantification of cyst nematodes), cyst isolation is required before DNA extraction. However, when isolating cysts from the soil, various impurities are simultaneously isolated, and separating the cysts from these impurities is laborious. Although previous studies have reported methods for extracting DNA from mixtures of cysts and impurities, it is unclear whether such DNA can be used to estimate nematode densities using qPCR. To examine the effects of impurities on the accuracy of qPCR quantification, we extracted DNA from nematode eggs (G. pallida and G. rostochiensis) mixed with impurities and performed qPCR. The results suggested that the differences in the fields affected the quantification accuracy. Therefore, field-specific standard curves should be set, which are impractical for routine diagnosis. To propose a more practical method, we determined a fixed standard curve for each species and estimated the population densities in field soil samples by qPCR using the standard curves. The estimated population densities significantly correlated with those determined using conventional microscopic inspections. This study revealed that the population densities of G. pallida and G. rostochiensis can be estimated from large amounts of soil, probably only approximately, but efficiently, by qPCR using DNA extracted from mixtures of cysts and impurities.This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1007/s13355-024-00863-ySpringer Nature’s AM terms of use;
https://www.springernature.com/gp/open-science/policies/accepted-manuscript-termsPublished: 24 February 2024journal articl
The collection and search sites in Kikaijima, Kakeromajima and Amami Oshima islands, Kagoshima prefecture.
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The first accession of Kakeromajima’s Vigna reflexo-pilosa (KKA15) for NARO Genebank with a yellow flower at the riverside of a small river running through Shodon Nagahama beach. The location is marked as KKA15 on the map (Fig. 1).
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Yellow flowers of Vigna luteola (KKA22?24), which are the northernmost accessions in Japan for NARO Genebank.
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