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

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.

CpGV-M Replication in Type I Resistant Insects: Helper Virus and Order of Ingestion Are Important.

The genetic diversity of baculoviruses provides a sustainable agronomic solution when resistance to biopesticides seems to be on the rise. This genetic diversity promotes insect infection by several genotypes (i.e., multiple infections) that are more likely to kill the host. However, the mechanism and regulation of these virus interactions are still poorly understood. In this article, we focused on baculoviruses infecting the codling moth, Cydia pomonella: two Cydia pomonella granulovirus genotypes, CpGV-M and CpGV-R5, and Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV). The influence of the order of ingestion of the virus genotypes, the existence of an ingestion delay between the genotypes and the specificity of each genotype involved in the success of multiple infection were studied in the case of Cydia pomonella resistance. To obtain a multiple infection in resistant insects, the order of ingestion is a key factor, but the delay for ingestion of the second virus is not. CrpeNPV cannot substitute CpGV-R5 to allow replication of CpGV-M.

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

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.

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

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.

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

To test the importance of the host genotype in maintaining virus genetic diversity, five experimental populations were constructed by mixing two Cydiapomonella 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.

Morphological properties of the occlusion body of Adoxophyes orana granulovirus.

A granulovirus (GV) that produces occlusion bodies (OBs) having an unusual morphology was found in an Adoxophyes sp. (Lepidoptera: Tortricidae) larva in a tea field in Miyazaki Prefecture, Japan. This isolate is considered to be a mutant of Adoxophyes orana granulovirus, designated AdorGV-M, because the nucleotide sequence of its genome is 99.7% identical to that of an English isolate of AdorGV, AdorGV-E. AdorGV-E produces typical ovocylindrical OBs that contain one occlusion-derived virus (ODV) per OB. On the other hand, AdorGV-M produces large cuboidal OBs, but the number of ODVs per OB was unknown. In this study, we quantified viral DNA in OBs of both AdorGV-E and -M, and determined the number of ODVs occluded in an OB of AdorGV-M. The two isolates had the same quantity of viral DNA in each OB, and we thus confirmed that one OB of AdorGV-M contains one ODV. To investigate the process of OB formation, fat body tissue of A. honmai larvae inoculated with each isolate was observed in a time course by transmission electron microscopy, and OB sizes were measured from micrographs. The main difference in OB formation was that AdorGV-M required more time to mature than AdorGV-E. In AdorGV-E, ODVs began to be covered from one end with an ovocylindrical OB at 96 h post-inoculation (hpi), and most of them were completely occluded at 120 hpi. Occlusion of AdorGV-M ODVs also began at 96 hpi, but the OB shape was cuboidal. Moreover, the OB size of AdorGV-M was similar to that of AdorGV-E at 120 hpi, but continued to grow until 192 hpi. AdorGV-M thus took more time to complete OB formation. Consequently, AdorGV-E has mature OBs with a diameter 0.22 µm and length 0.39 µm, but those of AdorGV-M are 1.34 × 1.23 µm.

Agrotis segetum nucleopolyhedrovirus but not Agrotis segetum granulovirus replicate in AiE1611T cell line of Agrotisipsilon.

Both Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV) belong to a cluster of four baculoviruses that are infective for different Agrotis species. Belonging further to different baculovirus genera, namely Alphabaculovirus and Betabaculovirus, respectively, AgseNPV-B and AgseGV are candidates to investigate virus interactions in co-infections. However, for the investigation of virus interactions on a cellular level, permissive insect cell-lines are needed. The cell line AiE1611T deriving from Agrotisipsilon eggs has been shown to be permissive for several Alphabaculovirus isolates. In this study, virus replication was followed based on microscopic analysis of infected and transfected cells, as well as on a molecular level by PCR of DNA and cDNA of selected baculovirus transcripts. While the permissivity was not verified for AgseGV, AgseNPV-B produced occlusion bodies in both infection with hemolymph of infected larvae and Lipofectamin transfection with AgseNPV-B genomic DNA. In addition to the possibility to investigate virus interaction of AgseNPV-B with other alphabaculoviruses, the permissivity of AiE1611T for AgseNPV-B further offers the possibility a biological selection to separate AgseNPV-B from AgseGV.

Genome Analysis and Genetic Stability of the Cryptophlebia leucotreta Granulovirus (CrleGV-SA) after 15 Years of Commercial Use as a Biopesticide.

Thaumatotibia leucotreta Meyrick (Lepidoptera: Tortricidae) is an indigenous pest in southern Africa which attacks citrus fruits and other crops. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme incorporating the baculovirus Cryptophlebialeucotreta granulovirus (CrleGV-SA) as a biopesticide has been implemented. This study investigated the genetic stability of a commercially produced CrleGV-SA product that has been applied in the field since 2000. Seven representative full-genome sequences of the CrleGV-SA isolate spanning a 15-year period were generated and compared with one another. Several open reading frames (ORFs) were identified to have acquired single nucleotide polymorphisms (SNPs) during the 15-year period, with three patterns observed and referred to as "stable", "reversion", and "unstable switching". Three insertion events were also identified, two of which occurred within ORFs. Pairwise multiple alignments of these sequences showed an identity ranging from 99.98% to 99.99%. Concentration-response bioassays comparing samples of CrleGV-SA from 2000 and 2015 showed an increase in virulence toward neonate T. leucotreta larvae. The CrleGV-SA genome sequence generated from the 2015 sample was compared to the Cape Verde reference genome, CrleGV-CV3. Several fusion events were identified between ORFs within these genomes. These sequences shared 96.7% pairwise identity, confirming that CrleGV-SA is a genetically distinct isolate. The results of this study indicate that the genome of CrleGV-SA has remained stable over many years, with implications for its continued use as a biopesticide in the field. Furthermore, the study describes the first complete baculovirus genome to be sequenced with the MinION (Oxford Nanopore, Oxford, UK) platform and the first complete genome sequence of the South African CrleGV isolate.

Using Next Generation Sequencing to Identify and Quantify the Genetic Composition of Resistance-Breaking Commercial Isolates of Cydia pomonella Granulovirus.

The use of Cydia pomonella granulovirus (CpGV) isolates as biological control agents of codling moth (CM) larvae is important in organic and integrated pome fruit production worldwide. The commercially available isolates CpGV-0006, CpGV-R5, and CpGV-V15 have been selected for the control of CpGV resistant CM populations in Europe. In infection experiments, CpGV-0006 and CpGV-R5 were able to break type I resistance and to a lower extent also type III resistance, whereas CpGV-V15 overcame type I and the rarely occurring type II and type III resistance. The genetic background of the three isolates was investigated with next generation sequencing (NGS) tools by comparing their nucleotide compositions to whole genome alignments of five CpGV isolates representing the known genetic diversity of the CpGV genome groups A to E. Based on the distribution of single nucleotide polymorphisms (SNPs) in Illumina sequencing reads, we found that the two isolates CpGV-0006 and CpGV-R5 have highly similar genome group compositions, consisting of about two thirds of the CpGV genome group E and one third of genome group A. In contrast, CpGV-V15 is composed of equal parts of CpGV genome group B and E. According to the identified genetic composition of these isolates, their efficacy towards different resistance types can be explained and predictions on the success of resistance management strategies in resistant CM populations can be made.

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

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.

Novel resistance to Cydia pomonella granulovirus (CpGV) in codling moth shows autosomal and dominant inheritance and confers cross-resistance to different CpGV genome groups.

Commercial Cydia pomonella granulovirus (CpGV) products have been successfully applied to control codling moth (CM) in organic and integrated fruit production for more than 30 years. Since 2005, resistance against the widely used isolate CpGV-M has been reported from different countries in Europe. The inheritance of this so-called type I resistance is dominant and linked to the Z chromosome. Recently, a second form (type II) of CpGV resistance in CM was reported from a field population (NRW-WE) in Germany. Type II resistance confers reduced susceptibility not only to CpGV-M but to most known CpGV isolates and it does not follow the previously described Z-linked inheritance of type I resistance. To further analyze type II resistance, two CM strains, termed CpR5M and CpR5S, were generated from parental NRW-WE by repeated mass crosses and selection using the two isolates CpGV-M and CpGV-S, respectively. Both CpR5M and CpR5S were considered to be genetically homogeneous for the presence of the resistance allele(s). By crossing and backcrossing experiments with a susceptible CM strain, followed by resistance testing of the offspring, an autosomal dominant inheritance of resistance was elucidated. In addition, cross-resistance to CpGV-M and CpGV-S was detected in both strains, CpR5M and CpR5S. To test the hypothesis that the autosomal inheritance of type II resistance was caused by a large interchromosomal rearrangement involving the Z chromosome, making type I resistance appear to be autosomal in these strains; fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC-FISH) was used to physically map the Z chromosomes of different CM strains. Conserved synteny of the Z-linked genes in CpR5M and other CM strains rejects this hypothesis and argues for a novel genetic and functional mode of resistance in CM populations with type II resistance.

Evidence for a Second Type of Resistance against Cydia pomonella Granulovirus in Field Populations of Codling Moths.

Cydia pomonella granulovirus (CpGV) is an important biocontrol agent for the codling moth (CM) in organic and integrated apple production worldwide. Previously, Z chromosome-linked dominant resistance in at least 38 CM field populations in Europe was reported, threatening organic apple production. Growers responded by switching to a different resistance-breaking isolate of CpGV that could control these populations. Here, we report a nonuniform response of different CM field populations to CpGV isolates from CpGV genome groups A to E. Even more strikingly, one field population, NRW-WE, was resistant to all known CpGV genome groups except group B. Single-pair crossing experiments with a susceptible strain, followed by resistance testing of the F1 offspring, clearly indicated cross-resistance to CpGV isolates that had been considered to be resistance breaking. This finding provides clear evidence of a second, broader type of CpGV resistance with a novel mode of inheritance that cannot be fully explained by Z-linkage of resistance. IMPORTANCE: CpGV is registered and used in virtually all commercial apple growing areas worldwide and is therefore the most widely used baculovirus biocontrol agent. Recently, resistance to CpGV products was reported in different countries in Europe, threatening organic growers who rely almost exclusively on CpGV products. This resistance appeared to be targeted against a 24-bp repeat in the pe38 gene in isolate CpGV-M of genome group A, which had been used commercially for many years. On the other hand, resistance could be broken by CpGV isolates from CpGV genome groups B to E. Here, we report clear evidence of a second type of field resistance that is also directed against resistance-breaking isolates of CpGV genome groups C, D, and E and which appears not to be targeted against CpGV pe38 Therefore, we propose to differentiate between type I resistance, which is targeted against pe38 of CpGV genome group A, and a novel type II resistance with an unknown molecular target. This finding stresses the need for further adoption of resistance management strategies for CpGV, since growers cannot rely solely on the use of resistance-breaking CpGV isolates.

A Betabaculovirus-Encoded gp64 Homolog Codes for a Functional Envelope Fusion Protein.

The GP64 envelope fusion protein is a hallmark of group I alphabaculoviruses. However, the Diatraea saccharalis granulovirus genome sequence revealed the first betabaculovirus species harboring a gp64 homolog (disa118). In this work, we have shown that this homolog encodes a functional envelope fusion protein and could enable the infection and fusogenic abilities of a gp64-null prototype baculovirus. Therefore, GP64 may complement or may be in the process of replacing F protein activity in this virus lineage.

Synergistic Effect of Combining Plutella xylostella Granulovirus and Bacillus thuringiensis at Sublethal Dosages on Controlling of Diamondback Moth (Lepidoptera: Plutellidae).

Plutella xylostella granulovirus (PxGV) and Bacillus thuringiensis (Bt) are both entomo-pathogens to the diamondback moth, Plutella xylostella (L.). The purpose of the present study was to measure the effect of the combination of PxGV and Bt at sublethal dosages on the development and mortality of diamondback moth in a laboratory setting. Bt and PxGV exhibited synergistic effect on diamondback moth larval mortality and effectively controlled diamondback moth populations with low dose combination treatment. The combination of three parts per million Bt and 1.3 × 10(3) occlusion bodies per milliliter of PxGV revealed a higher larval mortality compared with the treatment of Bt or PxGV alone. Combination of Bt and PxGV at sublethal concentrations also increased larval duration, reduced oviposition and decreased adult longevity remarkably, resulting in the lowest population trend index among the treatments. The results suggested that the combination of Bt and PxGV at sublethal dosages might provide a valuable way to improve the control efficacy of diamondback moth compared with treatment of Bt or PxGV alone.

Improving the Performance of the Granulosis Virus of Codling Moth (Lepidoptera: Tortricidae) by Adding the Yeast Saccharomyces cerevisiae with Sugar.

Studies were conducted with the codling moth granulosis virus (CpGV) to evaluate whether adding the yeast Saccharomyces cerevisiae Meyen ex E. C. Hansen with brown cane sugar could improve larval control of Cydia pomonella (L.). Larval mortalities in dipped-apple bioassays with S. cerevisiae or sugar alone were not significantly different from the water control. The addition of S. cerevisiae but not sugar with CpGV significantly increased larval mortality compared with CpGV alone. The combination of S. cerevisiae and sugar with CpGV significantly increased larval mortality compared with CpGV plus either additive alone. The addition of S. cerevisiae improved the efficacy of CpGV similarly to the use of the yeast Metschnikowia pulcherrima (isolated from field-collected larvae). The proportion of uninjured fruit in field trials was significantly increased with the addition of S. cerevisiae and sugar to CpGV compared with CpGV alone only in year 1, and from the controls in both years. In comparison, larval mortality was significantly increased in both years with the addition of S. cerevisiae and sugar with CpGV compared with CpGV alone or from the controls. The numbers of overwintering larvae on trees was significantly reduced from the control following a seasonal program of CpGV plus S. cerevisiae and sugar. The addition of a microencapsulated formulation of pear ester did not improve the performance of CpGV or CpGV plus S. cerevisiae and sugar. These data suggest that yeasts can enhance the effectiveness of the biological control agent CpGV, in managing and maintaining codling moth at low densities.

Mortality of cutworm larvae is not enhanced by Agrotis segetum granulovirus and Agrotis segetum nucleopolyhedrovirus B coinfection relative to single infection by either virus.

Mixed infections of insect larvae with different baculoviruses are occasionally found. They are of interest from an evolutionary as well as from a practical point of view when baculoviruses are applied as biocontrol agents. Here, we report mixed-infection studies of neonate larvae of the common cutworm, Agrotis segetum, with two baculoviruses, Agrotis segetum nucleopolyhedrovirus B (AgseNPV-B) and Agrotis segetum granulovirus (AgseGV). By applying quantitative PCR (qPCR) analysis, coinfections of individual larvae were demonstrated, and occlusion body (OB) production within singly infected and coinfected larvae was determined in individual larvae. Mixtures of viruses did not lead to changes in mortality rates compared with rates of single-virus treatments, indicating an independent action within host larvae under our experimental conditions. AgseNPV-B-infected larvae showed an increase in OB production during 2 weeks of infection, whereas the number of AgseGV OBs did not change from the first week to the second week. Fewer OBs of both viruses were produced in coinfections than in singly infected larvae, suggesting a competition of the two viruses for larval resources. Hence, no functional or economic advantage could be inferred from larval mortality and OB production from mixed infections of A. segetum larvae with AgseNPV-B and AgseGV.

Isolation of an Adoxophyes orana granulovirus (AdorGV) occlusion body morphology mutant: biological activity, genome sequence and relationship to other isolates of AdorGV.

A granulovirus (GV) producing occlusion bodies (OBs) with an unusual appearance was isolated from Adoxophyes spp. larvae in the field. Ultrastructural observations revealed that its OBs were significantly larger and cuboidal in shape, rather than the standard ovo-cylindrical shape typical of GVs. N-terminal amino acid sequence analysis of the OB matrix protein from this virus suggested that this new isolate was a variant of Adoxophyes orana granulovirus (AdorGV). Bioassays of this GV (termed AdorGV-M) and an English isolate of AdorGV (termed AdorGV-E) indicated that the two isolates were equally pathogenic against larvae of Adoxophyes honmai. However, AdorGV-M retained more infectivity towards larvae after irradiation with UV light than did AdorGV-E. Sequencing and analysis of the AdorGV-M genome revealed little sequence divergence between this isolate and AdorGV-E. Comparison of selected genes among the two AdorGV isolates and other Japanese AdorGV isolates revealed differences that may account for the unusual OB morphology of AdorGV-M.

Progressive adaptation of a CpGV isolate to codling moth populations resistant to CpGV-M.

The NPP-R1 isolate of CpGV is able to replicate on CpGV-M-resistant codling moths. However, its efficacy is not sufficient to provide acceptable levels of control in natural (orchard) conditions. A laboratory colony derived from resistant codling moths was established, which exhibited a homogeneous genetic background and a resistance level more than 7000 fold. By successive cycles of replication of NPP-R1 in this colony, we observed a progressive increase in efficacy. After 16 cycles (isolate 2016-r16), the efficacy of the virus isolate was equivalent to that of CpGV-M on susceptible insects. This isolate was able to control both CpGV-M-susceptible and CpGV-M-resistant insects with similar efficacy. No reduction in the levels of occlusion body production in susceptible larvae was observed for 2016-r16 compared to CpGV-M.

A single sex-linked dominant gene does not fully explain the codling moth's resistance to granulovirus.

BACKGROUND: In 2004, resistance to a commercial formulation of the Cydia pomonella granulovirus (CpGV) was identified in a field population of Cydia pomonella from an organic orchard in southern France. The genetic inheritance of this resistance was analysed in the resistant laboratory strain RGV. This strain was obtained using successive crosses between the resistant field population and a susceptible laboratory strain, SV, with selection for CpGV resistance at each generation. RESULTS: After eight generations of introgression of the resistant trait into SV, the RGV-8 strain exhibited 7000-fold higher resistance than SV. Mass-crossing experiments showed that resistance to CpGV is strongly dominant, sex dependent and under the control of a single major gene. However, the contribution of other genes is required to explain all of the data obtained in this study. These additional genes do not follow the laws of classical Mendelian transmission. CONCLUSION: Transmission of granulovirus resistance in the RGV-8 strain of C. pomonella cannot be fully explained by the effect of a locus located on the Z chromosome. The action of other factors needs to be considered.

Combining mutualistic yeast and pathogenic virus--a novel method for codling moth control.

The combination of a pathogenic virus and mutualistic yeasts isolated from larvae of codling moth Cydia pomonella is proposed as a novel insect control technique. Apples were treated with codling moth granulovirus (CpGV) and either one of three yeasts, Metschnikowia pulcherrima, Cryptococcus tephrensis, or Aureobasidium pullulans. The combination of yeasts with CpGV significantly increased mortality of neonate codling moth larvae, compared with CpGV alone. The three yeasts were equally efficient in enhancing the activity of CpGV. The addition of brown cane sugar to yeast further increased larval mortality and the protection of fruit against larvae. In comparison, without yeast, the addition of sugar to CpGV did not produce a significant effect. A field trial confirmed that fruit injury and larval survival were significantly reduced when apple trees were sprayed with CpGV, M. pulcherrima, and sugar. We have shown earlier that mutualistic yeasts are an essential part of codling moth larval diet. The finding that yeast also enhances larval ingestion of an insect-pathogenic virus is an opportunity for the development of a novel plant protection technique. We expect the combination of yeasts and insect pathogens to essentially contribute to future insect management.