The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei

Fouad Mokrini; Lieven Waeyenberge; Nicole Viaene; Fouad Abbad Andaloussi; Maurice Moens

doi:10.1163/15685411-00002808

The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei

in Nematology

Authors: Fouad Mokrini^1
,
2
,
3, Lieven Waeyenberge², Nicole Viaene^2
,
4, Fouad Abbad Andaloussi¹ and Maurice Moens^2
,
3

View More View Less

¹ National Institute of Agronomic Research (INRA), Km 9, 14000 Kenitra, Morocco

² Institute for Agricultural and Fisheries Research, Plant, Crop Protection, Burg. Van Gansberghelaan 96, B-9820 Merelbeke, Belgium

³ Laboratory for Agrozoology, Ghent University, Coupure links 653, B-9000 Ghent, Belgium

⁴ Department of Biology, Ghent University, K.L. Ledeganckstraat 35, B-9000 Ghent, Belgium

Online Publication Date:: 04 Sep 2014

Volume/Issue:: Volume 16: Issue 7

Article Type:: Research Article

DOI:: https://doi.org/10.1163/15685411-00002808

Keywords:: detection; lesion nematodes; plant-parasitic nematodes; Pratylenchidae; qPCR; sensitivity

Restricted Access

Get Access to Full Text

Get Permissions

A real-time quantitative PCR assay was developed for the accurate detection and quantification of the root-lesion nematode, Pratylenchus thornei. A qPCR primer set, including two primers and a probe, was designed based on the sequence of the β-1,4-endoglucanase gene. The assay was optimised by using the primers with SYBR green I dye and setting the qPCR program to different annealing temperatures ranging from 62 to 69°C. Based on the Ct values, we retained the program with an annealing temperature of 69°C. The specificity of the qPCR assay including the probe was confirmed by the lack of amplification of DNA from 47 populations belonging to 15 other Pratylenchus species and nine isolates from P. thornei. The assay was very sensitive as it was able to detect a single individual of P. thornei, even when mixed with up to 80 individuals of P. penetrans. DNA was extracted from exactly 80 P. thornei individuals. A dilution series from this DNA resulted in a standard curve showing a highly significant linearity between the Ct values and the dilution rates ( $R^{2} = 0.98$ ; slope = −3.38; $E = 97.6 %$ ). The qPCR assay developed in this study proved to be specific and sensitive, thus providing a fast and accurate tool for detection and quantification of this pathogen during research, as well as for diagnostic labs.

The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei

in Nematology

E-ISSN:: 1568-5411

Print ISSN:: 1388-5545

Publisher:: BRILL

Sections

References

Al-BannaL.PloegA.T.WilliamsonV.M.KaloshianI. (2004). Discrimination of six Pratylenchus species using PCR and species-specific primers. Journal of Nematology 36142-146.

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 22168-176.

Carrasco-BallesterosS.CastilloP.AdamsB.J.Pérez ArtésE. (2007). Identification of Pratylenchus thornei, the cereal and legume root-lesion nematode, based on SCAR-PCR and satellite DNA. European Journal of Plant Pathology 118115-125.

CastilloP.VovlasN. (2007). Pratylenchus (Nematoda: Pratylenchidae): diagnosis biology pathogenicity and management. Nematology Monographs and Perspectives 6 (Series editors: HuntD.J.PerryR.N.). Leiden, The NetherlandsBrill.

CiancioA.LoffredoA.PaeadiesF.TurturoC.SialerM.F. (2005). Detection of Meloidogyne incognita on Musa in Martinique, Guadeloupe, and French Guiana. Journal of Nematology 37313-322.

De LucaF.FanelliE.Di VitoM.ReyesA.De GiorgiC. (2004). Comparison of the sequences of the D3 expansion of the 26S ribosomal genes reveals different degrees of heterogeneity in different populations and species of Pratylenchus from the Mediterranean region. European Journal of Plant Pathology 111949-957.

De LucaF.ReyesA.TroccoliA.CastilloP. (2011). Molecular variability and phylogenetic relationships among different species and populations of Pratylenchus (Nematoda: Pratylenchidae) as inferred from the analysis of the ITS rDNA. European Journal of Plant Pathology 130415-426.

Di VitoM.GrecoN.SaxenaM.C. (1992). Pathogenicity of Pratylenchus thornei on chickpea in Syria. Nematologia Mediterranea 2071-73.

GlazerI.OrionD. (1983). Studies on anhydrobiosis of Pratylenchus thornei. Journal of Nematology 15333-338.

GrecoN.Di VitoM.SaxenaM.C.ReddyM.V. (1984). Investigation on the root lesion nematode Pratylenchus thornei in Syria. Nematologia Mediterranea 16101-105.

GrecoN.Di VitoM.SaxenaM.C. (1992). Plant parasitic nematodes of cool season food legumes in Syria. Nematologia Mediterranea 2037-46.

HendrickxG. (1995). An automated apparatus for extracting free-living nematode stages from soil. Nematologica 41308.

HoltermanM.van der WurffA.van den ElsenS.van MegenH.BongersT.HolovachovO.BakkerJ.HelderJ. (2006). Phylum-wide analysis of SSU rDNA reveals deep phylogenetic relationships among nematodes and accelerated evolution toward crown clades. Molecular Biology and Evolution 231792-1800.

LoofP.A.A. (1991). The family Pratylenchidae Thorne, 1949. In: NickleW.R. (Ed.). Manual of agricultural nematology. New York, NY, USAMarcel Dekker pp. 363-421.

LucM. (1987). A reappraisal of Tylenchina (Nemata). 7. The family Pratylenchidae Thorne, 1949. Revue de Nématologie 10203-218.

MadaniM.SubbotinS.A.MoensM. (2005). Quantitative detection of the potato cyst nematode, Globodera pallida and the beet cyst nematode, Heterodera schachtii, using real-time PCR with SYBR Green I dye. Molecular and Cellular Probes 1981-86.

MokriniF.WaeyenbergeL.ViaeneN.Abbad AndaloussiF.MoensM. (2013). Quantitative detection of the root-lesion nematode, Pratylenchus penetrans, using qPCR. European Journal of Plant Pathology 137403-413.

QiuJ.WesterdahlB.B.WilliamsonV.M. (2007). Detection and quantification of the root-lesion nematode Pratylenchus vulnus using real-time PCR. Journal of Nematology 3995-96.

SatoE.MinY.Y.SgirakashiT.WadaS.ToyotaK. (2007). Detection of the root-lesion nematode, Pratylenchus penetrans (Cobb), in a nematode community using real-time PCR. Journal of Nematology 3738-92.

SmileyR.W.WhittakerR.G.GourlieJ.A.EasleyS.A. (2005). Pratylenchus thornei associated with reduced wheat yield in Oregon. Journal of Nematology 3745-54.

SubbotinS.A.SturhanD.ChizhovV.N.VovlasN.BaldwinJ.G. (2006). Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology 8455-474.

ToyotaJ.B.ShirakashiT.SatoE.WadaS. (2008). Development of a real-time PCR method for the potato-cyst nematode Globodera rostochiensis and the root-knot nematode Meloidogyne incognita. Journal of Plant Nutrition and Soil Science 5472-76.

TroccoliA.LambertiF.GrecoN. (1992). Pratylenchus species occurring in Algeria (Nematoda, Pratylenchidae). Nematologia Mediterranea 2097-103.

UeharaT.MizukuboT.KushidaA.MomotaY. (1998). Identification of Pratylenchus penetrans (Cobb) by PCR using ITS-based specific. Japanese Journal of Nematology 281-7.

WaeyenbergeL.MoensM.PinochetJ.VrainT.C. (2000). Molecular characterisation of Pratylenchus species using rDNA restriction fragment length polymorphisms. Nematology 2135-142.

WaeyenbergeL.ViaeneN.MoensM. (2009). Species-specific duplex PCR for the detection of Pratylenchus penetrans. Nematology 11847-857.

YanG.P.SmileyR.W. (2013). Developing a real-time PCR assay for detection and quantification of Pratylenchus neglectus in soil. Plant Disease 97757-764.

YanG.P.SmileyR.W.OkubaraP.A.SkantarS.A.EasleyA.SheedyJ.G.ThompsonA.L. (2008). Detection and discrimination of Pratylenchus neglectus and P. thornei in DNA extracts from soil. Plant Disease 921480-1487.

YanG.P.SmileyR.W.OkubaraP.A. (2012). Detection and quantification of Pratylenchus thornei in DNA extracted from soil using real-time PCR. Phytopathology 10214-22.

Figures

View in gallery
A, B: Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 62°C and 63°C showing multiple peaks of two Pratylenchus thornei populations ( $n = 3$ ) and NTC ( $n = 2$ ); C, D: Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 65°C and 68°C showing multiple peaks of two P. thornei populations ( $n = 3$ ) and NTC ( $n = 2$ ).
View in gallery
Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 69°C showing high peaks at ±85.5 of two Pratylenchus thornei populations ( $n = 3$ ) and NTC ( $n = 2$ ).
View in gallery
Standard curve of the qPCR assay for Pratylenchus thornei threshold cycle number (Ct) plotted against the log of the number of individuals of P. thornei (1, 5, 10, 20, 40) ( $n = 3$ ).

A, B: Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 62°C and 63°C showing multiple peaks of two Pratylenchus thornei populations ( $n = 3$ ) and NTC ( $n = 2$ ); C, D: Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 65°C and 68°C showing multiple peaks of two P. thornei populations ( $n = 3$ ) and NTC ( $n = 2$ ).

Dissociation curve of the qPCR test (SensiFast SYBR Hi-ROX) with annealing temperatures set at 69°C showing high peaks at ±85.5 of two Pratylenchus thornei populations ( $n = 3$ ) and NTC ( $n = 2$ ).

Standard curve of the qPCR assay for Pratylenchus thornei threshold cycle number (Ct) plotted against the log of the number of individuals of P. thornei (1, 5, 10, 20, 40) ( $n = 3$ ).

Information

Page Count:: 789–796

Subjects:: Nematology, Biology

Copyright:: Copyright 2014 by Koninklijke Brill NV, Leiden, The Netherlands. 2014

Get Permissions

Content Metrics

	All Time	Past Year	Past 30 Days
Abstract Views	16	16	6
Full Text Views	67	67	48
PDF Downloads	4	4	2
EPUB Downloads	0	0	0

Recently viewed (1)

The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei

Article price

The β-1,4-endoglucanase gene is suitable for the molecular quantification of the root-lesion nematode, Pratylenchus thornei

Sections

References

Figures

Information

Content Metrics

Content Metrics