Quantification of Paratrichodorus allius in DNA extracted from soil using TaqMan Probe and SYBR Green real-time PCR assays

in Nematology
Restricted Access
Get Access to Full Text
Rent on DeepDyve

Have an Access Token?



Enter your access token to activate and access content online.

Please login and go to your personal user account to enter your access token.



Help

Have Institutional Access?



Access content through your institution. Any other coaching guidance?



Connect

The ectoparasitic stubby root nematode, Paratrichodorus allius, transmits tobacco rattle virus, which causes corky ringspot disease resulting in significant economic losses in the potato industry. A diagnostic method for direct quantification of P. allius from soil DNA using TaqMan probe and SYBR Green real-time PCR assays was developed to assist the potato industry in management of this important vector. Specificity of primers/probe designed from the internal transcribed spacer of ribosomal DNA of P. allius was demonstrated by in silico analysis and experimental PCR tests with no cross reactions using non-target nematode species and nematode communities. The SYBR Green method was more sensitive than the TaqMan probe method during detection using serial diluted DNA templates. Standard curves were generated from serial dilutions of DNA extracted from autoclaved soil with artificially inoculated P. allius individuals and were validated by high correlations between the numbers of target nematodes quantified by the assays and added to the soil. Moreover, the numbers of P. allius determined by the real-time PCR assays and estimated by the microscopic method in 17 field soil samples presented positive correlation relationships (R2=0.80 and 0.86). Although the quantification using TaqMan probe overestimated the target nematodes compared to using SYBR Green in eight out of ten field soil samples, results of the two methods correlated well (R2=0.92). This is the first report of P. allius quantification from soil DNA extracts using real-time PCR, providing a rapid and sensitive diagnostic method obviating time-consuming manual nematode extraction from soil and microscopic identification and quantification.

Nematology

International Journal of Fundamental and Applied Nematological Research

Sections

References

Al-BannaL.WilliamsonV.GardnerS.L. (1997). Phylogenetic analysis of nematodes of the genus Pratylenchus using nuclear 26 S rDNA. Molecular Phylogenetics and Evolution 7, 94-102. DOI: 10.1006/mpev.1996.0381

BerryS.D.FargetteM.SpaullV.W.MorandS.CadetP. (2008). Detection and quantification of root-knot nematode (Meloidogyne javanica), lesion nematode (Pratylenchus zeae) and dagger nematode (Xiphinema elongatum) parasites of sugarcane using real-time PCR. Molecular and Cellular Probes 22, 168-176. DOI: 10.1016/j.mcp.2008.01.003

BoutsikaK.PhillipsM.S.MacFarlaneS.A.BrownD.J.F.HolevaR.C.BlokV.C. (2004). Molecular diagnostics of some trichodorid nematodes and associated Tobacco rattle virus. Plant Pathology 53, 110-116. DOI: 10.1046/j.1365-3059.2003.00938.x

CaoH.ShockeyJ.M. (2012). Comparison of TaqMan and SYBR Green qPCR methods for quantitative gene expression in Tung tree tissues. Journal of Agricultural and Food Chemistry 60, 12296-12303. DOI: 10.1021/jf304690e

CherryT.SzalanskiA.T.ToddT.C.PowersT.O. (1997). The internal transcribed spacer region of Belonolaimus (Nemata: Belonolaimidae). Journal of Nematology 29, 23-29.

CiancioA.LoffredoA.ParadiesF.TurturoC.Finetti-SialerM. (2005). Detection of Meloidogyne incognita and Pochonia chlamydosporia by fluorogenic molecular probes. EPPO Bulletin 35, 157-164. DOI: 10.1111/j.1365-2338.2005.00794.x

CurranJ.BaillieD.L.WebsterJ.M. (1985). Use of restriction fragment length differences in genomic DNA to identify nematode species. Parasitology 90, 137-144. DOI: 10.1017/S0031182000049088

DecraemerW. (1995). The family Trichodoridae. Stubby root and virus vector nematodes. Dordrecht, The Netherlands, Kluwer Academic Publishers.

DecraemerW.BaujardP. (1998). A polytomous key for the identification of species of the family Trichodoridae Thorne, 1935 (Nematoda: Triplonchida). Fundamental and Applied Nematology 21, 37-62.

DecraemerW.CoolenW.HendrickxG.J. (1979). Evaluation of extraction methods Trichodoridae in fields of seed potatoes. Nematologica 25, 494-495. DOI: 10.1163/187529279X00640

HarrisonJ.M.GreenC.D. (1976). Comparison of centrifugal and other methods for standardization of extraction of nematodes from soil. Annals of Applied Biology 82, 299-308. DOI: 10.1111/j.1744-7348.1976.tb00565.x

HolevaR.PhillipsM.S.NeilsonR.BrownD.J.F.YoungV.BoutsikaK.BlokV.C. (2006). Real-time PCR detection and quantification of vector trichodorid nematodes and Tobacco rattle virus. Molecular and Cellular Probes 20, 203-211. DOI: 10.1016/j.mcp.2005.12.004

HollandP.M.AbramsonR.D.WatsonR.GelfandD.H. (1991). Detection of specific polymerase chain reaction product by utilizing the 53 exonuclease activity of Thermus aquaticus DNA polymerase. Proceedings of the National Academy of Sciences of the United States of America 88, 7276-7280.

HuangD.YanG.P.SkantarA.M. (2017). Development of real-time and conventional PCR assays for identifying stubby root nematode Paratrichodorus allius. Plant Disease 101, 964-972. DOI: 10.1094/PDIS-10-16-1431-RE

InghamR.E.HammP.B.BauneM.MerrifieldK.J. (2007). Control of Paratrichodorus allius and corky ringspot disease in potato with shank-injected metam sodium. Journal of Nematology 39, 258-262.

JenkinsW.R. (1964). A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48, 692.

KirkW.W.GieckS.L.CrosslinJ.M.HammP.B. (2008). First report of corky ringspot caused by Tobacco rattle virus on potatoes (Solanum tuberosum) in Michigan. Plant Disease 92, 485. DOI: 10.1094/PDIS-92-3-0485B

KumariS.SubbotinS.A. (2012). Molecular characterization and diagnostics of stubby root and virus vector nematodes of the family Trichodoridae (Nematoda: Triplonchida) using ribosomal RAN genes. Plant Pathology 61, 1021-1031. DOI: 10.1111/j.1365-3059.2012.02598.x

Lopez-NicoraH.D.MeketeT.SekoraN.NiblackT.L. (2014). First report of the stubby-root nematode (Paratrichodorus allius) from a corn field in Ohio. Plant Disease 98, 1164. DOI: 10.1094/PDIS-11-13-1180-PDN

MaiW.MullinP.G. (1996). Plant-parasitic nematodes: a pictorial key to genera, 5th edition. Ithaca, NY, USA, Cornell University Press.

MojtahediH.SantoG.S.HandooZ.CrosslinJ.M.BrownC.R.ThomasP.E. (2000). Distribution of Paratrichodorus allius and tobacco rattle virus in Pacific northwest potato fields. Journal of Nematology 32, 447.

MokriniF.WaeyenbergeL.ViaeneN.AndaloussiF.A.MoensM. (2013). Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR. European Journal of Plant Pathology 137, 403-413. DOI: 10.1007/s10658-013-0252-1

NegaA. (2014). Review on nematode molecular diagnostics: from bands to barcodes. Journal of Biology, Agriculture and Healthcare 4, 129-153. DOI: 10.1146/annurev.phyto.42.040803.140348

OkubaraP.A.SchroederK.L.PaulitzT.C. (2008). Identification and quantification of Rhizoctonia solani and R. oryzae using real-time polymerase chain reaction. Phytopathology 98, 837-847. DOI: 10.1094/PHYTO-98-7-0837

OliveiraC.M.G.MonteiroR.A.BlokV.C. (2011). Morphological and molecular diagnostics for plant-parasitic nematodes: working together to get the identification done. Tropical Plant Pathology 36, 65-73. DOI: 10.1590/S1982-56762011000200001

OliveiraC.M.G.BlokV.NeilsonR.MrózT.RobertsD. (2017). Hydrolysis probe-based PCR for detection of Pratylenchus crenatus, P. neglectus and P. penetrans. Nematology 19, 81-91. DOI: 10.1163/15685411-00003033

PlaisanceA.YanG.P. (2015). Comparison of two nematode extraction techniques. In: Abstracts of the 54th annual meeting of the Society of Nematologists, East Lansing, MI, USA, p. 120. [Abstr.]

PloegA.T.BrownD.J.F. (1997). Trichodorid nematodes and their associated viruses. In: SantosM.S.N. de A.AbrantesI.M. de O.BrownD.J.F.LemosR.M. (Eds). An introduction to virus vector nematodes and their associated viruses. Coimbra, Portugal, Institute do Ambiente e Vida, Universidade de Coimbra, pp.  41-68.

RigaE.KaranastasiE.OliveiraC.M.G.NeilsonR. (2007). Molecular identification of two stubby root nematode species. American Journal of Potato Research 84, 161-167. DOI: 10.1007/BF02987139

RigaE.LarsenR.EastwellK.GuerraN.GuerraL.CrosslinJ.M. (2009). Rapid detection of Tobacco Rattle Tobravirus in viruliferous Paratrichodorus allius from greenhouse and field specimens. Journal of Nematology 41, 60-63.

SatoE.GotoK.MinY.Y.ToyotaK.SuzukiC. (2010). Quantitative detection of Pratylenchus penetrans from soil using soil compaction and real-time PCR. Nematological Research 40, 1-6. DOI: 10.3725/jjn.40.1

SchneebergerC.SpeoserP.KuryF.ZeillingerR. (1995). Quantitative detection of reverse transciptase-PCR products by mean of a novel and sensitive DNA stain. PCR Methods and Applications 4, 234-238.

SubbotinS.A.MadaniM.KrallE.SturhanD.MoensM. (2005). Molecular diagnostics, taxonomy, and phylogeny of the stem nematode Ditylenchus dipsaci species complex based on the sequences of the internal transcribed spacer-rDNA. Phytopathology 95, 1308-1315. DOI: 10.1094/PHYTO-95-1308

TaylorC.E.BrownD.J.F. (1997). Nematode vectors of plant viruses. Wallingford, UK, CAB International.

YanG.P.SmileyR.W.OkubaraP.A. (2012). Detection and quantification of Pratylenchus thornei in DNA extracted from soil using real-time PCR. Phytopathology 102, 14-22. DOI: 10.1094/PHYTO-03-11-0093

YanG.P.SmileyR.W.OkubaraP.A.SkantarA.M.ReardonC.L. (2013). Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Disease 97, 757-764. DOI: 10.1094/PDIS-08-12-0729-RE

YanG.P.PlaisanceA.HuangD.UpadhayaA.GudmestadN.C.HandooZ.A. (2016). First report of the stubby root nematode Paratrichodorus allius on potato in North Dakota. Plant Disease 100, 1247. DOI: 10.1094/PDIS-11-15-1350-PDN

Figures

  • Standard curves of serial five-fold dilutions of soil DNA representing an equivalent of 20 to 2.56 × 10−4 individuals in 0.5 g of autoclaved soil. The circle and diamond markers represent individual values for each dilution and replicate. The black and dashed lines present the linear regression generated by TaqMan probe and SYBR Green real-time PCR, respectively.

    View in gallery
  • Relationship between the numbers of Paratrichodorus allius nematodes quantified by real-time PCR assays according to the generated standard curves and the numbers added to 0.5 g of autoclaved soil (0.5, 1, 2, 5, 10 and 15).

    View in gallery
  • Conventional PCR using universal primers rDNA2/rDNA1.58S for detection of DNA presence in the DNA extracts of potato field soil samples in Table 4. M = 100 bp DNA ladder. N = negative control which DNA was extracted from autoclaved soils. S1-S17 represent the DNA extracted from field samples 1-17. 1, 2 and 3 indicates the biological replicates for each soil DNA. H2O = no-template control using ddH2O instead of DNA in a PCR reaction. P = positive control of pure Paratrichodorus allius DNA.

    View in gallery
  • Relationship between the numbers of Paratrichodorus allius nematodes quantified by real-time PCR assays according to the generated standard curves and the numbers determined by manual nematode extraction and microscope method in 200 g of soil from potato field samples.

    View in gallery
  • Relationship between the numbers of Paratrichodorus allius nematodes quantified by TaqMan probe and SYBR Green real-time PCR assays.

    View in gallery

Information

Content Metrics

Content Metrics

All Time Past Year Past 30 Days
Abstract Views 12 12 6
Full Text Views 6 6 6
PDF Downloads 0 0 0
EPUB Downloads 0 0 0