Abstract
Real-time quantitative polymerase chain reaction (qPCR) is 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.
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The data supporting the findings of this study are available from the corresponding author upon reasonable request.
References
Gamel S, Letort A, Fouville D, Folcher L, Grenier E (2017) Development and validation of real-time PCR assays based on novel molecular markers for the simultaneous detection and identification of Globodera pallida, G. rostochiensis and Heterodera schachtii. Nematology 19:789–804. https://doi.org/10.1163/15685411-00003086
Goto K, Sato E, Toyota K (2009) A novel detection method for the soybean cyst nematode Heterodera glycines using soil compaction and real-time PCR. Nematol Res 39:1–7. https://doi.org/10.3725/jjn.39.1
Goto K, Sato E, Gang LF, Toyota K, Sugito T (2010) Comparison of calibration curves prepared by soil compaction and ball milling methods for direct quantification of the potato cyst nematode Globodera rostochiensis in soil. Nematol Res 40:41–45. https://doi.org/10.3725/jjn.40.41
Ikeda S, Watanabe KN, Minamisawa K, Ytow N (2004) Evaluation of soil DNA from arable land in Japan using a modified direct-extraction method. Microbes Environ 19:301–309. https://doi.org/10.1264/jsme2.19.301
Kushida A, Narabu T (2017) The sterilizing effects of heat or ethanol treatment on cysts of potato cyst nematodes. Ann Rept Plant Prot North Japan 68:150–154. https://doi.org/10.1145/kitanihon.2017.68_150
Kushida A, Sakai H (2022) Development of a simple method for simultaneous detection and differentiation of Globodera pallida and G. rostochiensis. J Gen Plant Pathol 88:251–258. https://doi.org/10.1007/s10327-022-01065-6
MacMillan K, Blok V, Young I, Crawford J, Wilson MJ (2006) Quantification of the slug parasitic nematode Phasmarhabditis hermaphrodita from soil samples using real time qPCR. Int J Parasitol 36:1453–1461. https://doi.org/10.1016/j.ijpara.2006.08.005
Madani M, Ward LJ, De Boer SH (2008) Multiplex real-time polymerase chain reaction for identifying potato cyst nematodes, Globodera pallida and Globadera rostochiensis, and the tobacco cyst nematode, Globadera tabacum. Can J Plant Pathol 30:554–564. https://doi.org/10.1080/07060660809507555
Min YY, Toyota K, Goto K, Sato E, Mizuguchi S, Abe N, Nakano A, Sawada E (2011) Development of a direct quantitative detection method for Meloidogyne incognita in sandy soils and its application to sweet potato cultivated fields in Tokushima prefecture, Japan. Nematology 13:95–102. https://doi.org/10.1163/138855410X504916
Nakhla MK, Owens KJ, Li W, Wei G, Skantar AM, Levy L (2010) Multiplex real-time PCR assays for the identification of the potato cyst and tobacco cyst nematodes. Plant Dis 94:959–965. https://doi.org/10.1094/PDIS-94-8-0959
Narabu T, Ohki T, Onodera K, Fujimoto T, Ito K, Maoka T (2016) First report of the pale potato cyst nematode, Globodera pallida, on potato in Japan. Plant Dis 100:1794. https://doi.org/10.1094/PDIS-12-15-1515-PDN
Papayiannis LC, Christoforou M, Markou YM, Tsaltas D (2013) Molecular typing of cyst-forming nematodes Globodera pallida and G. rostochiensis, using real-time PCR and evaluation of five methods for template preparation. J Phytopathol 161:459–469. https://doi.org/10.1111/jph.12091
R Development Core Team (2023) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. https://www.R-project.org/. Accessed 3 September 2023
Reid A, Evans F, Mulholland V, Cole Y, Pickup J (2015) High-throughput diagnosis of potato cyst nematodes in soil samples. In: Lacomme C (ed) Plant pathology: techniques and protocols, methods in molecular biology. Humana Press, New York, pp 137–148
Sakata I, Kushida A, Tanino K (2021a) The hatching-stimulation activity of solanoeclepin A toward the eggs of Globodera (Tylenchida: Heteroderidae) species. Appl Entomol Zool 56:51–57. https://doi.org/10.1007/s13355-020-00707-5
Sakata I, Sakai H, Kushida A (2021b) Subspecies identification of the Japanese population of Globodera tabacum. Nematol Res 51:37–40. https://doi.org/10.3725/jjn.51.37
Sakata I, Kushida A, Toyota K (2023) Species-specific detection of viable Globodera pallida using real-time reverse transcription PCR. Nematology 25:411–426. https://doi.org/10.1163/15685411-bja10228
Takada-Hoshino Y, Matsumoto N (2004) An improved DNA extraction method using skim milk from soils that strongly adsorb DNA. Microbes Environ 19:13–19. https://doi.org/10.1264/jsme2.19.13
Trudgill DL, Evans K, Parrott DM (1975a) Effects of potato cyst nematodes on potato plants. I. Effects in a trial with irrigation and fumigation on the growth and nitrogen and potassium contents of a resistant and a susceptible variety. Nematologica 21:169–182
Trudgill DL, Evans K, Parrott DM (1975b) Effects of potato cyst nematodes on potato plants. II. Effects on haulm size, concentration of nutrients in haulm tissue and tuber yield of a nematode resistant and a nematode susceptible potato variety. Nematologica 21:183–191
van den Elsen S, Ave M, Schoenmakers N, Landeweert R, Bakker J, Helder J (2012) A rapid, sensitive, and cost-efficient assay to estimate viability of potato cyst nematodes. Phytopathology 102:140–146. https://doi.org/10.1094/PHYTO-02-11-0051
Volossiouk T, Robb EJ, Nazar RN (1995) Direct DNA extraction for PCR-mediated assays of soil organisms. Appl Environ Microbiol 61:3972–3976. https://doi.org/10.1128/aem.61.11.3972-3976.1995
Yamada E, Takakura S, Tezuka H (1972) On the occurrence of the potato cyst nematode, Heterodera rostochiensis Wollenweber in Hokkaido, Japan. Jpn J Nematol 2:12–15. https://doi.org/10.14855/jjn1972.2.12
Acknowledgements
We thank Mr. Katsuyuki Morita and Mr. Mitsuyuki Tsujiguchi (JA Youtei) for their help with the soil sampling. We thank Dr. Kenji Asano, Dr. Hiromichi Sakai, and Mr. Gaku Murata (National Agriculture and Food Research Organization) for providing soil samples derived from Memuro, Tsukuba, and Koshi, respectively. This study was supported by the Development and Improvement Program of Strategic Smart Agricultural Technology Grant (JPJ011397) from the Project of the Bio-Oriented Technology Research Advancement Institution.
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Conceptualization: Itaru Sakata, Kenji Itou, and Atsuhiko Kushida; sample collection and investigation: Itaru Sakata and Kenji Itou; writing of the original draft: Itaru Sakata; and review and editing: Itaru Sakata, Kenji Itou, and Atsuhiko Kushida.
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No approval from the research ethics committees was required to accomplish the goals of this study because the experimental work was conducted with an unregulated invertebrate species.
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Sakata, I., Itou, K. & Kushida, A. Efficient quantification of Globodera pallida and G. rostochiensis (Tylenchida: Heteroderidae) in large amounts of soil using probe-based real-time PCR. Appl Entomol Zool 59, 145–153 (2024). https://doi.org/10.1007/s13355-024-00863-y
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DOI: https://doi.org/10.1007/s13355-024-00863-y