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

Viruses ◽  
2019 ◽  
Vol 11 (8) ◽  
pp. 723 ◽  
Author(s):  
Aurélie Hinsberger ◽  
Stéphane Theulier Saint Germain ◽  
Patrice Guerrero ◽  
Christine Blachère-López ◽  
Miguel López-Ferber ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Cydia Pomonella ◽  
Relative Proportion ◽  
Variable Region ◽  
Codling Moth ◽  
Pcr Primers ◽  
Type I ◽  
Occlusion Bodies ◽  
Type I Resistance

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.

scholarly journals Importance of the Host Phenotype on the Preservation of the Genetic Diversity in Codling Moth Granulovirus

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 621 ◽  
Author(s):  
Graillot ◽  
Blachere-López ◽  
Besse ◽  
Siegwart ◽  
López-Ferber
Keyword(s):  
Genetic Diversity ◽  
Cydia Pomonella ◽  
Codling Moth ◽  
Type I ◽  
Host Genotype ◽  
Host Colony ◽  
Type I Resistance ◽  
Cydia Pomonella Granulovirus ◽  
Marker Virus ◽  
Mexican Isolate

To test the importance of the host genotype in maintaining virus genetic diversity, five experimental populations were constructed by mixing two Cydia pomonella granulovirus isolates, the Mexican isolate CpGV-M and the CpGV-R5, in ratios of 99% M + 1% R, 95% M + 5% R, 90% M + 10% R, 50% M + 50% R, and 10% M + 90% R. CpGV-M and CpGV-R5 differ in their ability to replicate in codling moth larvae carrying the type I resistance. This ability is associated with a genetic marker located in the virus pe38 gene. Six successive cycles of replication were carried out with each virus population on a fully-permissive codling moth colony (CpNPP), as well as on a host colony (RGV) that carries the type I resistance, and thus blocks CpGV-M replication. The infectivity of offspring viruses was tested on both hosts. Replication on the CpNPP leads to virus lineages preserving the pe38 markers characteristic of both isolates, while replication on the RGV colony drastically reduces the frequency of the CpGV-M pe38 marker. Virus progeny obtained after replication on CpNPP show consistently higher pathogenicity than that of progeny viruses obtained by replication on RGV, independently of the host used for testing.

scholarly journals Partial Loss of Inheritable Type I Resistance of Codling Moth to Cydia pomonella granulovirus

Viruses ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 570 ◽  
Author(s):  
Jiangbin Fan ◽  
Jörg Wennmann ◽  
Johannes Jehle
Keyword(s):  
Current Knowledge ◽  
Full Range ◽  
Cydia Pomonella ◽  
Codling Moth ◽  
Single Pair ◽  
Type I ◽  
Partial Loss ◽  
Resistance Level ◽  
Type I Resistance ◽  
Cydia Pomonella Granulovirus

Current knowledge of the field resistance of codling moth (CM, Cydia pomonella, L) against Cydia pomonella granulovirus (CpGV) is based mainly on the interaction between the Mexican isolate CpGV-M and CpRR1, a genetically homogeneous CM inbreed line carrying type I resistance. The resistance level of laboratory-reared CpRR1 to CpGV-M was recently found to have decreased considerably, compared to the initially high resistance. To understand the background of this phenomenon, CpRR1 larvae were exposed over several generations to CpGV-M for re-selection of the original resistance level. After five and seven generations of selection, new CpRR1_F5 and CpRR1_F7 lines were established. The resistance ratio of these selected lines was determined by full range bioassays. The CpRR1_F5 strain regained a higher level of resistance against CpGV up to 104-fold based on LC50 values compared to susceptible larvae (CpS), which indicated that the absence of virus selection had resulted in a reduction of resistance under laboratory rearing conditions. In addition, some fitness costs of fecundity were observed in CpRR1_F5. Single-pair crossings between CpRR1_F5 or CpRR1_F7 with susceptible CpS moths revealed a dominant but not fully sex-linked inheritance, which suggests a partial loss of previous resistance traits in CpRR1.

scholarly journals Transcriptome of Cydia pomonella granulovirus in susceptible and type I resistant codling moth larvae

2021 ◽  
Vol 102 (3) ◽  
Author(s):  
Jörg T. Wennmann ◽  
Diana Pietruska ◽  
Johannes A. Jehle
Keyword(s):  
Gene Expression ◽  
Fat Body ◽  
Cydia Pomonella ◽  
Codling Moth ◽  
Viral Gene ◽  
Type I ◽  
Pome Fruit ◽  
Type I Resistance ◽  
Cydia Pomonella Granulovirus

The baculovirus Cydia pomonella granulovirus (CpGV) is a biocontrol agent used worldwide against the codling moth (CM), Cydia pomonella L., a severe pest in organic and integrated pome fruit production. Its successful application is increasingly challenged by the occurrence of CM populations resistant to commercial CpGV products. Whereas three types (I–III) of CpGV resistance have been identified, type I resistance compromising the efficacy of CpGV-M, the so-called Mexican isolate of CpGV, is assumed to be the most widely distributed resistance type in Central Europe. Despite the wide use of CpGV products as biocontrol agents, little information is available on gene-expression levels in CM larvae. In this study, the in vivo transcriptome of CpGV-M infecting susceptible (CpS) and resistant (CpRR1) CM larvae was analysed at 24, 48, 72, 96 and 120 hours post infection in the midgut and fat body tissue by using a newly developed microarray covering all ORFs of the CpGV genome. According to their transcript abundance, the CpGV genes were grouped into four temporal clusters to which groups of known and unknown function could be assigned. In addition, sets of genes differentially expressed in the midgut and fat body were found in infected susceptible CpS larvae. For the resistant CpRR1 larvae treated with CpGV-M, viral entry in midgut cells could be confirmed from onset but a significantly reduced gene expression, indicating that type I resistance is associated with a block of viral gene transcription and replication.

scholarly journals Novel Diversity and Virulence Patterns Found in New Isolates of Cydia pomonella Granulovirus from China

2019 ◽  
Vol 86 (2) ◽  
Author(s):  
Jiangbin Fan ◽  
Jörg T. Wennmann ◽  
Dun Wang ◽  
Johannes A. Jehle
Keyword(s):  
Biocontrol Agent ◽  
Control Strategies ◽  
Cydia Pomonella ◽  
Codling Moth ◽  
Future Application ◽  
Type I ◽  
Resistance Breaking ◽  
Control Options ◽  
Type I Resistance ◽  
Cydia Pomonella Granulovirus

ABSTRACT Cydia pomonella granulovirus (CpGV) is successfully used worldwide as a biocontrol agent of the codling moth (CM) (Cydia pomonella). The occurrence of CM populations with different modes of resistance against commercial CpGV preparations in Europe, as well as the invasiveness of CM in China, threatening major apple production areas there, requires the development of new control options. Utilizing the naturally occurring genetic diversity of CpGV can improve such control strategies. Here, we report the identification of seven new CpGV isolates that were collected from infected CM larvae in northwest China. Resistance testing using a discriminating CpGV concentration and the determination of the median lethal concentration (LC50) were performed to characterize their levels of virulence against susceptible and resistant CM larvae. The isolates were further screened for the presence of the 2 × 12-bp-repeat insertion in CpGV gene pe38 (open reading frame 24 [ORF24]), which was shown to be the target of type I resistance. It was found that three isolates, CpGV-JQ, -KS1, and -ZY2, could break type I resistance, although delayed mortality was observed in the infection process. All isolates followed the pe38 model of breaking type I resistance, except for CpGV-WW, which harbored the genetic factor but failed to overcome type I resistance. However, CpGV-WW was able to overcome type II and type III resistance. The bioassay results and sequencing data of pe38 support previous findings that pe38 is the major target for type I resistance. The new isolates show some distinct virulence characteristics when infection of different CM strains is considered. IMPORTANCE CpGV is a highly virulent pathogen of the codling moth (CM). It is registered and widely applied as a biocontrol agent in nearly all apple-growing countries worldwide. The emergence of CpGV resistance and the increasing lack of chemical control options require improvements to current control strategies. Natural CpGV isolates, as well as resistance-breaking isolates selected in resistant CM strains, have provided resources for improved resistance-breaking CpGV products. Here, we report novel CpGV isolates collected in China, which have new resistance-breaking capacities and may be an important asset for future application in the biological control of codling moths.

scholarly journals Genome Analysis of A Novel South African Cydia pomonella granulovirus (CpGV-SA) with Resistance-Breaking Potential

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 658 ◽  
Author(s):  
Boitumelo Motsoeneng ◽  
Michael D. Jukes ◽  
Caroline M. Knox ◽  
Martin P. Hill ◽  
Sean D. Moore
Keyword(s):  
South African ◽  
Complete Genome ◽  
Cydia Pomonella ◽  
Codling Moth ◽  
Type I ◽  
Nucleotide Polymorphisms ◽  
Pome Fruit ◽  
Single Nucleotide ◽  
Resistance Breaking ◽  
Cydia Pomonella Granulovirus

The complete genome of an endemic South African Cydia pomonella granulovirus isolate was sequenced and analyzed. Several missing or truncated open reading frames (ORFs) were identified, including a 24 bp deletion in the pe38 gene which is reported to be associated with type I resistance-breaking potential. Comparison of single nucleotide polymorphisms (SNPs) with five other fully sequenced CpGV isolates identified 67 unique events, 47 of which occurred within ORFs, leading to several amino acid changes. Further analysis of single nucleotide variations (SNVs) within CpGV-SA revealed that this isolate consists of mixed genotypes. Phylogenetic analysis using complete genome sequences placed CpGV-SA basal to M, I12 and E2 and distal to S and I07 but with no distinct classification into any of the previously defined CpGV genogroups. These results suggest that CpGV-SA is a novel and genetically distinct isolate with significant potential as a biopesticide for management of codling moth (CM), not only in South Africa, but potentially in other pome fruit producing countries, particularly where CM resistance to CpGV has been reported.

scholarly journals Snapback Primer Genotyping of the Gilbert Syndrome UGT1A1 (TA)n Promoter Polymorphism by High-Resolution Melting

2011 ◽  
Vol 57 (9) ◽  
pp. 1303-1310 ◽  
Author(s):  
Jared S Farrar ◽  
Robert A Palais ◽  
Carl T Wittwer
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Pcr Primers ◽  
Promoter Polymorphism ◽  
Fragment Analysis ◽  
Gilbert Syndrome ◽  
Blinded Study ◽  
Thymine Adenine ◽  
Local Deviation ◽  
Genotype Clustering

BACKGROUND Gilbert syndrome, a chronic nonhemolytic unconjugated hyperbilirubinemia, is associated with thymine–adenine (TA) insertions in the UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) promoter. The UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan. Current closed-tube assays for genotyping the UGT1A1 (TA)n promoter polymorphism require multiple labeled probes and/or have difficulty classifying the (TA)5 and (TA)8 alleles. METHODS An unlabeled 5′ extension on one primer that creates a hairpin after asymmetric PCR was used to develop a snapback primer high-resolution melting assay for the (TA)n polymorphism. A new method that plots the local deviation from exponential decay to improve genotype clustering was used to remove background fluorescence and to analyze the data. The snapback assay was compared with small-amplicon melting and fragment length analyses in a blinded study of DNA samples from 100 African Americans. RESULTS Genotyping results obtained by small-amplicon melting and snapback primer melting were 83% and 99% concordant, respectively, with results obtained by fragment analysis. Reanalysis of the single discordant sample in the results of the snapback genotyping assay and the fragment analysis revealed an error in the fragment analysis. High-resolution melting was required for accurate snapback genotyping of the UGT1A1 (TA)n polymorphism. The 100% accuracy obtained with a capillary-based instrument fell to ≤81% with plate-based instruments. CONCLUSIONS In contrast to small-amplicon genotyping, snapback primer genotyping can distinguish all UGT1A1 promoter genotypes. Rapid-cycle PCR combined with snapback primer analysis with only 2 unlabeled PCR primers (one with a 5′ extension) and a saturating DNA dye can genotype loci with several alleles in <30 min.

scholarly journals Amplicon Melting Analysis with Labeled Primers: A Closed-Tube Method for Differentiating Homozygotes and Heterozygotes

2003 ◽  
Vol 49 (3) ◽  
pp. 396-406 ◽  
Author(s):  
Cameron N Gundry ◽  
Joshua G Vandersteen ◽  
Gudrun H Reed ◽  
Robert J Pryor ◽  
Jian Chen ◽  
...  
Keyword(s):  
High Resolution ◽  
High Resolution Melting ◽  
Rapid Heating ◽  
Melting Curve ◽  
Pcr Primers ◽  
High Resolution Melting Analysis ◽  
Melting Transition ◽  
Sequence Variant ◽  
Sequence Variants ◽  
Melting Analysis

Abstract Background: Common methods for identification of DNA sequence variants use gel electrophoresis or column separation after PCR. Methods: We developed a method for sequence variant analysis requiring only PCR and amplicon melting analysis. One of the PCR primers was fluorescently labeled. After PCR, the melting transition of the amplicon was monitored by high-resolution melting analysis. Different homozygotes were distinguished by amplicon melting temperature (Tm). Heterozygotes were identified by low-temperature melting of heteroduplexes, which broadened the overall melting transition. In both cases, melting analysis required ∼1 min and no sample processing was needed after PCR. Results: Polymorphisms in the HTR2A (T102C), β-globin [hemoglobin (Hb) S, C, and E], and cystic fibrosis (F508del, F508C, I507del, I506V) genes were analyzed. Heteroduplexes produced by amplification of heterozygous DNA were best detected by rapid cooling (>2 °C/s) of denatured products, followed by rapid heating during melting analysis (0.2–0.4 °C/s). Heterozygotes were distinguished from homozygotes by a broader melting transition, and each heterozygote had a uniquely shaped fluorescent melting curve. All homozygotes tested were distinguished from each other, including Hb AA and Hb SS, which differed in Tm by <0.2 °C. The amplicons varied in length from 44 to 304 bp. In place of one labeled and one unlabeled primer, a generic fluorescent oligonucleotide could be used if a 5′ tail of identical sequence was added to one of the two unlabeled primers. Conclusion: High-resolution melting analysis of PCR products amplified with labeled primers can identify both heterozygous and homozygous sequence variants.

scholarly journals Cross-Resistance of the Codling Moth against Different Isolates of Cydia pomonella Granulovirus Is Caused by Two Different but Genetically Linked Resistance Mechanisms

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 1952
Author(s):  
Annette J. Sauer ◽  
Eva Fritsch ◽  
Karin Undorf-Spahn ◽  
Kento Iwata ◽  
Regina G. Kleespies ◽  
...  
Keyword(s):  
Biological Control Agent ◽  
Cydia Pomonella ◽  
Codling Moth ◽  
Resistance Mechanisms ◽  
Cross Resistance ◽  
Viral Gene ◽  
Type I ◽  
Type Ii ◽  
Cydia Pomonella Granulovirus ◽  
Type Ii Resistance

Cydia pomonella granulovirus (CpGV) is a widely used biological control agent of the codling moth. Recently, however, the codling moth has developed different types of field resistance against CpGV isolates. Whereas type I resistance is Z chromosomal inherited and targeted at the viral gene pe38 of isolate CpGV-M, type II resistance is autosomal inherited and targeted against isolates CpGV-M and CpGV-S. Here, we report that mixtures of CpGV-M and CpGV-S fail to break type II resistance and is expressed at all larval stages. Budded virus (BV) injection experiments circumventing initial midgut infection provided evidence that resistance against CpGV-S is midgut-related, though fluorescence dequenching assay using rhodamine-18 labeled occlusion derived viruses (ODV) could not fully elucidate whether the receptor binding or an intracellular midgut factor is involved. From our peroral and intra-hemocoel infection experiments, we conclude that two different (but genetically linked) resistance mechanisms are responsible for type II resistance in the codling moth: resistance against CpGV-M is systemic whereas a second and/or additional resistance mechanism against CpGV-S is located in the midgut of CpR5M larvae.

scholarly journals Cydia pomonella granulovirus Genotypes Overcome Virus Resistance in the Codling Moth and Improve Virus Efficiency by Selection against Resistant Hosts

2008 ◽  
Vol 75 (4) ◽  
pp. 925-930 ◽  
Author(s):  
Marie Berling ◽  
Christine Blachere-Lopez ◽  
Olivier Soubabere ◽  
Xavier Lery ◽  
Antoine Bonhomme ◽  
...  
Keyword(s):  
Genetic Characterization ◽  
Cydia Pomonella ◽  
Codling Moth ◽  
Resistance Level ◽  
Laboratory Colony ◽  
Occlusion Bodies ◽  
Insect Populations ◽  
Cydia Pomonella Granulovirus ◽  
First Time ◽  
Resistant Trait

ABSTRACT Cydia pomonella granulovirus (CpGV) has been used for 15 years as a bioinsecticide in codling moth (Cydia pomonella) control. In 2004, some insect populations with low susceptibility to the virus were detected for the first time in southeast France. RGV, a laboratory colony of codling moths resistant to the CpGV-M isolate used in the field, was established with collection of resistant insects in the field followed by an introgression of the resistant trait into a susceptible colony (Sv). The resistance level (based on the 50% lethal concentrations [LC50s]) of the RGV colony to the CpGV-M isolate, the active ingredient in all commercial virus formulations in Europe, appeared to be over 60,000-fold compared to the Sv colony. The efficiency of CpGV isolates from various other regions was tested on RGV. Among them, two isolates (I12 and NPP-R1) presented an increased pathogenicity on RGV. I12 had already been identified as effective against a resistant C. pomonella colony in Germany and was observed to partially overcome the resistance in the RGV colony. The recently identified isolate NPP-R1 showed an even higher pathogenicity on RGV than other isolates, with an LC50 of 166 occlusion bodies (OBs)/μl, compared to 1.36 � 106 OBs/μl for CpGV-M. Genetic characterization showed that NPP-R1 is a mixture of at least two genotypes, one of which is similar to CpGV-M. The 2016-r4 isolate obtained from four successive passages of NPP-R1 in RGV larvae had a sharply reduced proportion of the CpGV-M-like genotype and an increased pathogenicity against insects from the RGV colony.

scholarly journals A Third Type of Resistance to Cydia pomonella Granulovirus in Codling Moths Shows a Mixed Z-Linked and Autosomal Inheritance Pattern

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
A. J. Sauer ◽  
S. Schulze-Bopp ◽  
E. Fritsch ◽  
K. Undorf-Spahn ◽  
J. A. Jehle
Keyword(s):  
Biocontrol Agent ◽  
Cydia Pomonella ◽  
Codling Moth ◽  
Field Resistance ◽  
Type I ◽  
Pome Fruit ◽  
Content Type ◽  
Genome Group ◽  
Cydia Pomonella Granulovirus ◽  
Autosomal Inheritance

ABSTRACT Different isolates of Cydia pomonella granulovirus (CpGV) are used worldwide to control codling moth larvae (Cydia pomonella) in pome fruit production. Two types of dominantly inherited field resistance of C. pomonella to CpGV have been recently identified: Z-chromosomal type I resistance and autosomal type II resistance. In the present study, a CpGV-resistant C. pomonella field population (termed SA-GO) from northeastern Germany was investigated. SA-GO individuals showed cross-resistance to CpGV isolates of genome group A (CpGV-M) and genome group E (CpGV-S), whereas genome group B (CpGV-E2) was still infective. Crossing experiments between individuals of SA-GO and the susceptible C. pomonella strain CpS indicated the presence of a dominant autosomal inheritance factor. By single-pair inbreeding of SA-GO individuals for two generations, the genetically more homogenous strain CpRGO was generated. Resistance testing of CpRGO neonates with different CpGV isolates revealed that isolate CpGV-E2 and isolates CpGV-I07 and -I12 were resistance breaking. When progeny of hybrid crosses and backcrosses between individuals of resistant strain CpRGO and susceptible strain CpS were infected with CpGV-M and CpGV-S, resistance to CpGV-S appeared to be autosomal and dominant for larval survivorship but recessive when success of pupation of the hybrids was considered. Inheritance of resistance to CpGV-M, however, is proposed to be both autosomal and Z linked, since Z linkage of resistance was needed for pupation. Hence, we propose a further type III resistance to CpGV in C. pomonella, which differs from type I and type II resistance in its mode of inheritance and response to CpGV isolates from different genome groups. IMPORTANCE The baculovirus Cydia pomonella granulovirus (CpGV) is registered and applied as a biocontrol agent in nearly all pome fruit-growing countries worldwide to control codling moth caterpillars in an environmentally friendly manner. It is therefore the most widely used commercial baculovirus biocontrol agent. Since 2005, field resistance of codling moth to CpGV products has been observed in more than 40 field plantations in Europe, threatening organic and integrated apple production. Knowledge of the inheritance and mechanism(s) of resistance is indispensable for the understanding of host response to baculovirus infection on the population level and the coevolutionary arms race between virus and host, as well as for the development of appropriate resistance management strategies. Here, we report a codling moth field population with a new type of resistance, which appears to follow a highly complex inheritance in regard to different CpGV isolates.

Sign in / Sign up

Export Citation Format

Share Document