A combination of real-time PCR and high-resolution melting analysis to detect and identify CpGV genotypes involved in type I resistance

Aurélie Hinsberger (1, 2) , Stéphane Theulier Saint Germain (3) , Patrice Guerrero (3) , Christine Blachère-Lopez (4, 1, 2) , Miguel Lopez-Ferber (1, 2) , Sandrine Bayle (2)

1 UM - Université de Montpellier
2 LGEI - Laboratoire de Génie de l'Environnement Industriel
3 Ecole de l’ADN
4 DPT SPE - Département Santé des Plantes et Environnement

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.

Mots clés: high resolution melting (HRM); biological control agents biological control organisms; pe38 gene; resistance gene; detection method; orchard; Cydia pomonella granulovirus; codling moth; pomology; weed control methods; biological control; resistance

Mots clés: culture fruitière; méthode de lutte; carpocapse; gène de résistance; méthode de détection; agent de lutte biologique; verger; pathologie végétale; comportement de résistance

Domaines: Sciences agricoles; Biologie animale; Phytopathologie et phytopharmacie

Aurélie Hinsberger, Stéphane Theulier Saint Germain, Patrice Guerrero, Christine Blachère-Lopez, Miguel Lopez-Ferber, et al.. A combination of real-time PCR and high-resolution melting analysis to detect and identify CpGV genotypes involved in type I resistance. Viruses, 2019, 11 (8), pp.723. ⟨10.3390/v11080723⟩. ⟨hal-02310107⟩