A Combination of Real-Time PCR and High-Resolution Melting Analysis to Detect and Identify CpGV Genotypes Involved in Type I Resistance

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Aurélie Hinsberger 1, Stéphane Theulier Saint Germain 2, Patrice Guerrero 2, Christine Blachère-López 1,3, Miguel López-Ferber 1 and Sandrine Bayle 1,*
1
LGEI, Ecole des Mines d’Alès, Institut Mines-Télécom et Université de Montpellier Sud de France, 6 Avenue de Clavières, 30100 Alès, France
2
Ecole de l’ADN, 13 Boulevard Amiral Courbet, 30000 Nîmes, France
3
INRA, 6 Avenue de Clavières, 30319 Alès, France
*
Author to whom correspondence should be addressed.
Viruses 2019, 11(8), 723; https://doi.org/10.3390/v11080723
Received: 28 May 2019 / Revised: 26 July 2019 / Accepted: 2 August 2019 / Published: 6 August 2019
(This article belongs to the Special Issue Insect Viruses and Pest Management)

Abstract

Cydia pomonella granulovirus, in particular CpGV-M isolate, is used as a biological control against the codling moth (CM), Cydia pomonella. As a result of intensive control over the years, codling moth populations have developed resistance against this isolate. This resistance is now called type I resistance. Isolates, among them, CpGV-R5, have been found that are able to overcome type I resistance. Both CpGV-M and CpGV-R5 are used in orchards to control the codling moth. High resolution melting (HRM) has been adapted to differentiate between CpGV-M and CpGV-R5 isolates. Specific PCR primers have been designed for the CpGV p38 gene, encompassing the variable region responsible for the ability to overcome resistance. Because each amplicon has a specific melting point, it is possible to identify the CpGV-M and CpGV-R5 genotypes and to quantify their relative proportion. This method has been validated using mixtures of occlusion bodies of each isolate at various proportions. Then, the HRM has been used to estimate the proportion of each genotype in infected larvae or in occlusion bodies (OBs) extracted from dead larvae. This method allows a rapid detection of genotype replication and enables the assessment of either success or failure of the infection in field conditions. View Full-Text

Keywords: Cydia pomonella granulovirus; codling moth; biological control; resistance; high resolution melting (HRM); pe38 gene
Article on line viruses-11-00723