Genomic Structure And Virulence Difference Of Seven Isolates Of Cydia Pomonella Granulovirus

Cydia pomonella granulovirus?Cp GV?is a specific and 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 Cp GV as well as the existence of resistance-breaking Cp GV isolates have been discovered,providing an ideal model for studying baculovirus-host adaptation.This thesis aims to elucidate the virulence difference of recently collected isolates of Cp GV from northwest China when they were exposed to susceptible and resistant CM colonies,as well as their genomic polymorphisms.The potential adjuvant added into Cp GV was also studied.The population structure of seven Cp GV isolates was analyzed on the basis of Illumina next generation sequence?NGS?data.The isolates were termed as Cp GV-ZY,-JQ,-ALE,-KS1,-KS2,-ZY2 and-WW.First,It was found that the isolates Cp GV-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 Cp GV-WW,which lacked the 2×12 bp repeat involved in resistance-breaking but failed to overcome type I resistance.However,Cp GV-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 Cp RR1,except for Cp GV-WW.Second,After Illumina NGS sequencing,the genome assembly,annotation and phylogenetic analyses of these isolates indicated that the genomes were highly conserved and related to known Cp GV isolates,despite a considerable geographic distance.However,two new phylogenetic lineages,termed genome groups F?Cp GV-JQ and-ZY2?and G?Cp GV-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 of223 new SNP positions out of total 563 SNPs were detected against Cp GV-M reference sequence,which represented virus characteristics of Chinese isolates.Whereas Cp GV-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 Cp GV-ZY,-KS1 and-KS2 were highly similar and were composed of variable ratios of genome group A?Cp GV-M?and genome group E?Cp GV-WW?.Third,in laboratory and semi-field bioassays,the virulence and persistence of Cp GV-ZY were significant increase when mixed with 7mg/ml Fe₂O₃ in both bioassays.It also enhanced Cp GV-ZY adaptability to strong UV and sunlight.Compared to control of Cp GV-ZY,the mortality was raised 53.88%and 96.64%,respectively,in laboratory and field conditions.The virulence and persistence of Cp GV-ZY was markedly enhanced by adding low dose of Fe₂O₃,which showed that this compound was candidate adjuvents of Cp GV-ZY products and enhance virus efficiency against codling moth.Fourth,when third instar larvae of Cp S and Cp RR1 ingested Cp GV with the dose of LD₅₀,the transcription level of heat shock protein 70 was investigated.In constrast to untreated control,the transcription levels of hsp70-1 and hsp70-2 of Cp S infected with Cp GV-M were first increased and then declined.On the constrary,the transcription levels of hsp70-1 and hsp70-2 of Cp RR1 infected with Cp GV-S were stable at the beginning of inection and suddenly increased more than three times at 7 dpi comparing to untreatment.In comparision of the hsp70s transcription phase in Cp S and Cp RR1 showed the transcription of this gene was delayed about two-day in Cp RR1 larvae.Successful Cp GV infection in codling moth larva was together with the increasing transcription of hsp70s.In summary,seven newly discovered Cp GV isolates exhibited variable resistance-breaking ability against known types of field resistance of CM,of which the highly pathogenic isolates can build up a virus resource for sustainable development of Cp GV agent in control of codling moth.The established methods to determine positional SNP distribution can be easily extended to other baculoviruses to assess isolate composition and genetic diversity.Low dose of Fe₂O₃ can protect Cp GV from the UV damage,showing the potential of becoming the ingredient of virus agent.The increasing transcription of hsp70s is correlated to resultful Cp GV infection in colding moth.Understanding the genetic adaption between baculovirus and host insect,and properly using anti-UV agent may give a crucial blueprint to improve current strategies of Cp GV resistance management and sustainable development in the field.