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.

Effects of a Covert Infection with Phthorimaea operculella granulovirus in Insect Populations of Phthorimaea operculella.

Virus infections of insects can easily stay undetected, neither showing typical signs of a disease, nor being lethal. Such a stable and most of the time covert infection with Phthorimaea operculella granulovirus (PhopGV) was detected in a Phthorimaea operculella laboratory colony, which originated from Italy (Phop-IT). This covert virus (named PhopGV-R) was isolated, purified and characterized at the genetic level by full genome sequencing. Furthermore, the insect colony Phop-IT was used to study the crowding effect, double infection with other PhopGV isolates (CR3 and GR1), and co-infection exclusion. An infection with a second homologous virus (PhopGV-CR3) activated the covert virus, while a co-infection with another virus isolate (PhopGV-GR1) led to its suppression. This study shows that stable virus infections can be common for insect populations and have an impact on population dynamics because they can suppress or enable co-infection with another virus isolate of the same species.

Interaction of Phthorimaea operculella granulovirus with a Nosema sp. microsporidium in larvae of Phthorimaea operculella.

An antagonistic effect of a microsporidium (Nosema sp.) infection on the virulence of Phthorimaea operculella granulovirus (PhopGV) was recorded in potato tuber moth (Phthorimaea operculella) larvae with mixed infections. When the P. operculella colony was infected at a high rate (42.8-100%) with the microsporidium, it was less susceptible to the isolate PhopGV-GR1.1. A virus concentration 1.89 × 105 higher was necessary to cause the same level of mortality produced in the P. operculella colony when it was uninfected or had a low level of infection with the microsporidium (0-30%). This antagonistic effect was driven by a Nosema isolate (termed Nosema sp. Phop) that was purified from microsporidian-infected P. operculella individuals. The purified microsporidium was characterised by morphological features, including size, filament coils and different developmental stages using transmission electron microscopy (TEM). On the molecular level, the partial cistron rDNA information of the small ribosomal subunit (SSU), internal transcribed spacer (ITS), and the large ribosomal subunit (LSU) were identified. Phylogenetic analyses revealed that the newly described microsporidium belongs to the "true Nosema" clade. Partial sequence information of the RNA polymerase II largest subunit (RPB1) suggested that Nosema bombycis is the closest relative (98% identity). The morphological and phylogenetic characteristics suggest that it is an isolate of N. bombycis. Interactions of microsporidia and betabaculoviruses are rarely described in the literature, although mixed infections of different pathogens seem to be rather common events, ranging from antagonistic to mutualistic interactions. The observed antagonistic relationship between the Nosema sp. and PhopGV-GR1.1 showed that pathogen interactions need to be considered when single pathogens are applied to insect populations in the context of biological control of insect pests.

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.

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.

High stability and no fitness costs of the resistance of codling moth to Cydia pomonella granulovirus (CpGV-M).

Resistance against the biocontrol agent Cydia pomonella granulovirus (CpGV-M) was previously observed in field populations of codling moth (CM, C. pomonella) in South-West Germany. Incidental observations in a laboratory reared field colony (CpR) indicated that this resistance is rather stable, even in genetically heterogeneous CM colonies consisting of both susceptible and resistant individuals. To test this hypothesis, the resistance level of CpR that was 1000times less susceptible to CpGV-M was followed for more than 60 generations of rearing. Even without virus selection pressure, the high level of resistance, expressed as median lethal concentration, remained stable for more than 30 generations and declined only by a factor of 10 after 60 generations. When cohorts of the F32 and F56 generations of the same colony were selected to CpGV-M for five and two generations, respectively, the resistance level increased to factor of >1,000,000 compared to a susceptible control colony. Laboratory reared colonies of CpR, did not exhibit any measurable fitness costs under laboratory conditions in terms of fecundity and fertility. Resistance testing of seven selected codling moth field populations collected between 2003 and 2008 in commercial orchards in Germany that were repeatedly sprayed with CpGV products gave evidence of different levels of resistance and a more than 20-fold increase of the resistance in 1-3 years when selection by CpGV-M was continued. A maximum 1,000,000-fold level of resistance to CpGV-M that could be induced in the laboratory under virus pressure had been also observed in one field population. The high stability of resistance observed in the genetically heterogenous colony CpR indicates that resistance to CpGV-M is not very costly.