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No description available.

No description available.

Graduation date: 1995

Bertha armyworm, Mamestra configurata (Lepidoptera: Noctuidae), is an important pest of cruciferous oilseed crops in western Canada. A nuclear polyhedrosis virus, MacoNPV, isolated from M. configurata, has demonstrated as high as 95% infection in field populations of bertha armyworm. MacoNPV isolates from different geographic areas differ in terms of their virulence to bertha armyworm. Restriction endonuclease (REN) fragment analyses show that all of the geographic isolates are closely related viruses but with some distinct REN pattern differences. Most of these geographic isolates are heterogenous mixtures of genotypes. The thesis describes the cloning and physical mapping of the 156.9 kbp genome of the MacoNPV-90/2 geographic isolate, including 112 restriction sites for six common REN, BamHI, EcoRI, HindIII, PstI, SmaI and XhoI. Twenty plaque purified strains of MacoNPV were isolated in a cultured Mamestra brassicae (Mbr) insect cell line. The EcoRI restriction patterns of these pick plaque (pp) strains fell into 10 different categories. In order to investigate the difference among these pp strains, and between these strains and the parental geographic isolates in terms of REN patterns, virulence to insect hosts, and their growth rates in insect cell lines, some of these isolates were selected for bioassays in bertha armyworm larvae and in the Mbr cell line.

Thesis (Ph. D.)--University of Adelaide, Dept. of Crop Protection, 1998? / Includes bibliographical references (leaves 120-145).

Bibliography: leaves 120-145. Reports Bt crystal protein toxicity to a phthirapteran species. Although Bt strain WB3516 may produce other unidentified toxins effective against B. ovis, the results provide strong evidence that the [delta]-endotoxin crystal proteins of strain WB3516 significantly contribute to the lousicidal toxicity of this strain.

Thesis (MSc (Biomedical Sciences))--Cape Peninsula University of Technology, 2019 / Background: Baculoviruses have great potential as alternatives to conventional chemical insecticides. The large scale adoption of such agents has however been hampered by the slow killing times exhibited by these bio-insecticides, limitation to single target insect and difficulty of large scale production of these preparations. Trichoplusia ni single nucleopolyhedrovirus (TnSNPV), initially identified in the Eastern Cape region of South Africa, has potential as a biocontrol agent as it possesses a higher speed of kill compared to other baculoviruses. Aims and methods: The main objective of this study was the identification, molecular characterisation and cloning of a structural core gene (polyhedrin) and three auxiliary genes, the inhibitor of apoptosis (iap2 and iap3) and the ecdysteroid UDP-glucosyltransferase (egt) genes, from TnSNPV in order to delineate its phylogenetic relationship to a Canadian isolate of the same virus and to other baculoviruses. In addition, the genes were expressed in an Escherichia coli (E. coli) based system as a prelude to genetic modification to increase the pesticidal property of the virus. Results: The genome size of the South African strain of TnSNPV was estimated at 160 kb and is significantly larger than the Canadian isolate of TnSNPV and may reflect genetic variation as the two strains have adapted to varying environmental conditions. Occlusion bodies of the South African strain of TnSNPV were visualised by Transmission Electron Microscopy and consisted of rod shaped single virions composed of a single enveloped nucleocapsid. Insect bioassays showed that the median lethal time (LT50) of the virus strain averaged 1.8 days which is significantly faster than other baculoviruses. The South African and Canadian strains of TnSNPV share nucleotide similarities greater than 95% for the genes analysed in this study, which indicates that they are closely related. From this analysis, the South African strain of TnSNPV identifies as a Group II NPV with the closest relatives being the Canadian strain of TnSNPV and ChchNPV. The topology of the tree for the polyhedrin protein was better resolved than that of the IAP2, IAP3 and EGT proteins and was comparable to the tree inferred from a concatenated data set consisting of complete polyhedrin/granulin, LEF8, and LEF9 proteins of 48 completely sequenced genomes. For the IAP2, IAP3 and EGT proteins, the separation of the lepidopteran and hymenopteran specific baculoviruses was not evident while the separation of Group I and II Alphabaculoviruses diverged from that observed from the baculovirus core gene polyhedrin as well as the tree inferred from complete polyhedrin/granulin, LEF8, and LEF9 proteins. Five distinct groups relating to IAP-1, 2, 3, 4 and 5 could be distinguished from the tree inferred from all IAP proteins from 48 fully sequenced baculoviruses. From this analysis, the IAP protein from the South African isolate of TnSNPV can be designated as an IAP3 due to sequence homology to other IAP3 proteins. Similarly, the IAP2 can be confirmed as an IAP2 protein as it clusters with other IAP2 proteins. RNA transcripts of the four genes were detected by RT-PCR at one hr after induction with Larabinose in BL21-A1 E. coli and persisted until four hrs post induction. Antisera directed against the C-terminal 6X His tag was able to detect the recombinant proteins at two hours after induction confirming the rapid rise in expression of the proteins which persisted at high levels until four hrs after induction. The discrepancy observed with the predicted molecular mass of the EGT protein and the migration on SDS-PAGE may be due to the absence of posttranslational modification in the E. coli expression system and the hydrophobic residues present in the N-terminal signal sequence. Conclusion: Sequence and phylogenetic analysis suggest that the two isolates of TnSNPV have been exposed to similar evolutionary pressures and evolved at similar rates and represent closely related but distinct variants of the same virus. The difference in genome size between the two strains is likely to reflect actual genetic differences as the strains have adapted to their local environments and hosts and the extent of the differences will only be apparent as more sequencing results become available. Phylogenetic analysis of the IAP and EGT proteins yields a tree that varies from the phylogenetic reconstruction observed for the polyhedrin gene as well as the concatenated data set consisting of complete polh/gran, LEF8, and LEF9 proteins and highlights the risks inherent in inferring phylogenetic relationships based on single gene sequences. The tree inferred from the concatenated data set of polh/gran, LEF8, and LEF9 proteins was a quick and reliable method of identification particularly, when whole genome data is unavailable and mirrors the accepted lineage of baculoviruses. Expression of the recombinant IAP2, IAP3, EGT and polyhedrin was confirmed by RT-PCR and immunoblot analysis and rose rapidly after induction and persisted at high levels. It is as yet unclear if the expressed proteins are functional particularly as post translation modifications are lacking in this system.

Bibliography: leaves 120-145. / vii, 145, [43] leaves, [23] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reports Bt crystal protein toxicity to a phthirapteran species. Although Bt strain WB3516 may produce other unidentified toxins effective against B. ovis, the results provide strong evidence that the [delta]-endotoxin crystal proteins of strain WB3516 significantly contribute to the lousicidal toxicity of this strain. / Thesis (Ph.D.)--University of Adelaide, Dept. of Crop Protection, 1998?

Baculoviruses are arthropod-specific DNA viruses that are highly virulent to most lepidopteran insects. Their host specificity and compatibility with IPM programmes has enabled their usage as safe microbial insecticides (biopesticides). Two baculovirus-based biopesticides, Cryptogran and Cryptex, which have been formulated with Cryptophlebia leucotreta granulovirus (CrleGV) have been registered for the control of false codling moth (FCM), Thaumatotibia (=Cryptophlebia) leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa and have been successfully incorporated into IPM programmes. However, several studies have indicated that insects can develop resistance to baculovirus-based biopesticide as was shown with field populations of codling moth (CM), Cydia pomonella (L.), which developed resistance to the biopesticide Cydia pomonella granulovirus (CpGV-M) in Europe. Other studies have shown that, under laboratory conditions, FCM populations differ in their susceptibility to Cryptogran and Cryptex. In order to investigate difference in susceptibility as well as protect against any future resistance by FCM to Cryptogran and Cryptex, a search for novel CrleGV-SA isolates from diseased insects from different geographic regions in South Africa was performed. Six geographic populations (Addo, Citrusdal, Marble Hall, Nelspruit, Baths and Mixed colonies) of FCM were established and maintained in the laboratory. Studies on the comparative biological performance based on pupal mass, female fecundity, egg hatch, pupal survival, adult eclosion and duration of life cycle of the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies revealed a low biological performance for the Citrusdal colony. This was attributed to the fact that FCM populations found in the Citrusdal area are not indigenous and may have been introduced from a very limited gene pool from another region. When insects from five colonies, excluding the Baths colony, were subjected to stress by overcrowding , a latent baculovirus resident in the Addo, Nelspruit, Citrusdal, Marble Hall and Mixed colonies was brought into an overt lethal state. Transmission electron micrographs revealed the presence of GV occlusion bodies (OBs) in diseased insects. DNA profiles obtained by single restriction endonuclease analysis of viral genomic DNA using BamH 1, Sa/1, Xba1 , Pst1, Xh01 , Kpn1, Hindlll and EcoR1 revealed five CrleGV-SA isolates latent within the insect populations. The new isolates were named CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl, CrleGVSA Nels and CrleGV-SA Mix isolates. The novelty of the five CrleGV-SA isolates was confirmed by the presence of unique submolar bands, indicating that each isolate was genetically different. PCR amplification and sequencing of the granulin and egt genes from the five isolates revealed several single nucleotide polymorph isms (SNPs) which, in some cases, resulted in amino acid substitutions. DNA profiles from RFLPs, as well as phylogenetic analysis based on granulin and egt sequencing showed the presence of two CrleGV-SA genome types for the CrleGV-SA isolate. Cryptex and CrleGV-SA Ado, CrleGV-SA Cit, CrleGV-SA Mbl and CrleGV-SA Mix were placed as members of Group one CrleGV-SA, and Cryptogran and CrleGV-SA Nels isolate were placed into Group two CrleGV-SA. In droplet feeding bioassays, the median survival time (STso) for neonate larvae inoculated with Group one and two CrleGV-SA were determined to range from 80 - 88 hours (3.33 - 3.67 days), for all five colonies. LDso values for Group one and two CrleGV-SA against neonates from the Addo, Citrusdal, Marble Hall, Nelspruit and Mixed colonies varied between some populations and ranged from 0.80 - 3.12 OBs per larva, indicating some level of variation in host susceptibility. This is the first study reporting the existence of genetically distinct CrleGV baculovirus isolates infecting FCM in different geographical areas of South Africa. The results of this study have broad-ranging implications for our understanding of baculovirus-host interactions and for the application of baculovirus basedbiopesticides.