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.

Different isolates of Cydia pomonella granulovirus (CpGV) are used worldwide to control codling moth larvae () in pome fruit production. Two types of dominantly inherited field resistance of to CpGV have been recently identified: Z-chromosomal type I resistance and autosomal type II resistance. In the present study, a CpGV-resistant 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 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, which differs from type I and type II resistance in its mode of inheritance and response to CpGV isolates from different genome groups. 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.

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.

The aim of this study is to establish a quantitative determination of the vegetative insecticidal protein Vip3A from the culture supernatant of Bacillus thuringiensis either by ELISA or by the conventional quantification method of the Western blot band. The Vip3A protein was produced by fermentation of the B. thuringiensis reference strain BUPM95 in 3 L. By Western blot, the Vip3Aa16 toxin was detected in the culture supernatant during the exponential growth phase of B. thuringiensis BUPM95. However, the detection of Vip3Aa16 on Western blot showed in addition to the toxin two other strips (62 and 180 kDa) recognized by the anti-Vip3Aa16 polyclonal antibodies prepared at the Centre of Biotechnology of Sfax Tunisia. For that reason and in order to develop a technique for reliable quantification of the toxin, we have considered the production of polyclonal antibodies at the Julius Kühn Institute, Germany. These antibodies were the basis for the production of monoclonal antibodies directed against the protein produced by the Vip3Aa16 recombinant strain Escherichia coli BL21 (DE3). These monoclonal antibodies were tested by plate-trapped antigen (PTA) and triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA). The selection of hybridoma supernatants gave us four positive clones producing monoclonal antibodies.

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.