Cydia pomonella granulovirus (CpGV) is a highly virulent pathogen of codling moth (CM, Cydia pomonella L.) larvae. It has been developed to one of the most successful commercial baculovirus biocontrol agents used on hundred thousands of hectares of pome fruit production worldwide. In recent years, however, three types (I to III) of field resistance to CpGV as well as the existence of resistance-breaking CpGV isolates have been discovered, providing an ideal model for studying baculovirus-host adaptation. This thesis aims to elucidate the potential of recently collected isolates of CpGV from northwest China to infect susceptible and resistant CM colonies and to study the stability and restoration of type I resistance in laboratory rearing by mass-crossing and selection. To further explore the genotypic and biological differences of CpGV, the population structure of 20 CpGV isolates was analyzed on the basis of Illumina next generation sequence (NGS) data. The isolates included seven new Chinese CpGV isolates, termed CpGV-ZY, -JQ, -ALE, -KS1, -KS2, -ZY2 and -WW, the re-sequenced isolates CpGV-M, -S, -E2, -I12 and the Iranian isolate CpGV-I0X, as well as the active ingredients of commercial virus selections from MadexPlus, MadexMAX, MadexTOP (V15), V14, V34, V45 and Carpovirusine EVO2.
First, resistance testing or full range bioassays were conducted to determine the resistance-breaking capacity or the median lethal concentration (LC50) of the Chinese CpGV isolates against susceptible and three resistant CM strains, representing type I to III resistance (Chapter 2). The isolates were further screened for the presence of the additional 2×12 bp repeat insertion in CpGV gene pe38 (ORF24), which had been proposed to be the target of type I resistance in the Mexican isolate CpGV-M. It was found that the isolates CpGV-JQ, -KS1 and -ZY2 could break type I resistance, though a distinct delay was observed in the infection process. The isolates followed the previously established “pe38 model” of resistance-breaking, except CpGV-WW, which lacked the 2×12 bp repeat involved in resistance-breaking but failed to overcome type I resistance. However, CpGV-WW was able to overcome type II and type III resistance. Correlation of bioassay results and the isolates’ pe38 repeat structure were in agreement with the potential role of pe38 as the major target for resistance in CpRR1, except for CpGV-WW.
Second, resistance tests with CpGV-M revealed a certain decline of the resistance level of CM strain CpRR1, expressing type I resistance, after it had been reared for several years without virus pressure (Chapter 3). Therefore, two newly selected lines, CpRR1_F5 and CpRR1_F7, were established by mass crossing experiments combined with virus selection on CpGV-M. Resistance level of the newly selected lines was determined by full range bioassays. The successful selection process resulted in a 15- to 160-fold increase of the LC50 of CpRR1_F5 resistance compared to CpRR1, suggesting that the rearing in absence of virus selection was most likely the main factor involved the observed resistance decline of CpRR1. Additionally, some fitness costs of fecundity were recorded in the re-selected CpRR1_F5. Single-pair crossing of CpRR1_F5 and CpRR1-F7 with susceptible CM, followed by a resistance testing with a discriminating concentration of CpGV-M occlusion bodies, revealed a dominant but not fully sex-linked inheritance arguing for a partial change of previous genetic traits in CpRR1.
Third, in Chapter 4 the genomic difference among seven new Chinese CpGV isolates could provide some answers for the virulence difference observed in bioassays. After Illumina NGS sequencing, the genome annotation and phylogenetic analyses of these isolates indicated that the genomes were highly conserved and related to known CpGV isolates, despite a considerable geographic distance. However, two new phylogenetic lineages, termed genome groups F (CpGV-JQ and -ZY2) and G (CpGV-ALE), were proposed in addition to previous phylogenetic genome groups A to E. The genetic composition of the isolates was further quantified on the basis of previously identified genome group specific single nucleotide polymorphisms (SNPs). In addition of 223 new SNP positions out of total 563 SNPs were detected against CpGV-M reference sequence, which represented virus characteristics of Chinese isolates. Whereas CpGV-WW was proposed to be genetically highly homogeneous, belonging to genome group E, the other six isolates were mixtures of at least two genotypes. Thereof CpGV-ZY, -KS1 and -KS2 were highly similar and were composed of variable ratios of genome group A (CpGV-M) and genome group E (CpGV-WW). Detailed quantification of the 12 bp repeat unit of pe38 corresponded to the results obtained from PCR and Sanger sequence analyses (Chapter 2).
Fourth, to achieve a fully comprehensive perspective of CpGVs of different origin, 20 CpGV isolates, including twelve natural isolates from different geographic locations and eight selected CpGV strains, were analyzed together for the distribution and frequency of single nucleotide polymorphisms (SNPs) in NGS genome data and for the abundance of the 12 bp repeat unit in pe38 (Chapter 5). The results indicated that CpGV-M, -WW, -S and MadexPlus were genetically highly homogenous isolates with a low rate of polymorphisms, while other isolates were composed of two or more genome groups at different ratios. Based on hierarchical clustering on principal components (HCPC) analysis, six distinct clusters were proposed, which represents the previously proposed main phylogenetic lineages, though the insertions and deletions were not included in cluster analysis. Relative location of different isolates in HCPC further reflected the ratio of variable compositions of different genome groups. For the quantification of the proportions of 1-5×12 bp repeat units in the different CpGV isolates a “read counting” method was developed and showed a high diversity and less conserved characteristics in pe38 than literature reported before. The established methods for SNP quantification and HCPC analysis provide novel tools to decipher the molecular complexity of genome mixtures in virus isolates, thus depicting the population structure of baculovirus isolates in a more adequate form than genome consensus based analyses.
In summary, the results in this thesis showed that resistance loss in CpRR1 is developing in laboratory under continuous rearing without virus pressure. Newly discovered CpGV isolates exhibited high potential for control of known types of field resistance of CM. The established methods to determine positional SNP distribution can be easily extended to other (baculo)viruses to assess isolate composition and genetic diversity and to study quality and stability of virus mixtures during propagation. It can be further applied to determine its potential for control of resistant CM on molecular level, since CpGV isolates with the similar virulence patterns were found to be grouped together considering their spatial location in factor map of HCPC. Understanding CpGV population structure and the genetic adaption between baculovirus and host insect give a crucial blueprint to improve current strategies of CpGV resistance management in the field
