Development of qPCR assays for quantitative detection of Heterodera avenae and H. latipons

Twelve Heterodera species are considered of major economic significance in cereals, of which Heterodera avenae, H. latipons and H. filipjevi are the most important. Precise identification and quantification of these nematodes are necessary to develop effective integrated pest control. This study rep...

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Bibliographic Details
Published in:European journal of plant pathology 2015-10, Vol.143 (2), p.305-316
Main Authors: Toumi, F, Waeyenberge, L, Viaene, N, Dababat, A. A, Nicol, J. M, Ogbonnaya, F. C, Moens, M
Format: Article
Language:English
Subjects:
Agriculture
assays
Biomedical and Life Sciences
Cereals
Crops
cytochrome-c oxidase
Deoxyribonucleic acid
DNA
DNA primers
Ecology
Eggs
genes
Grain
grains
Heterodera
Heterodera avenae
juveniles
Life Sciences
mixtures
Nematoda
Nematodes
Pest control
Plant Pathology
Plant Sciences
quantitative polymerase chain reaction
rapid methods
Studies
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Summary:Twelve Heterodera species are considered of major economic significance in cereals, of which Heterodera avenae, H. latipons and H. filipjevi are the most important. Precise identification and quantification of these nematodes are necessary to develop effective integrated pest control. This study reports on the use of the mitochondrial cytochrome oxidase subunit 1 (COI) gene to develop qPCR assays that could be used for the identification and quantification of H. avenae and H. latipons. Two qPCR primer sets, each comprising two primers and a probe, were designed for each of both species. After optimization, the qPCR assays using a single second-stage juvenile (J2) were able to identify and quantify H. avenae and H. latipons. Their specificity was confirmed by the lack of amplification of J2 of 14 other Heterodera species. A qPCR using DNA extracted from 120 J2 + eggs of H. avenae and H. latipons resulted in steady Ct-values (Ct = 22.33 ± 0.1 and Ct = 21.83 ± 0.12, respectively). Dilution series of DNA extracted from 120 J2 + eggs of the two species were made. The assays for both species resulted in a standard curve showing a highly significant linearity between the Ct-values and the dilution rates (R² = 0.99; slope = −3.03 and R² = 0.99; slope = −3.28 for H. avenae and H. latipons, respectively). The two qPCR assays provide a sensitive and valid tool for rapid detection and quantification of the two species whether they occur alone or in mixtures with other species.
ISSN:0929-1873
1573-8469
DOI:10.1007/s10658-015-0681-0