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The Isolation, Genetic Characterisation And The isolation, genetic characterisation and biological activity of a South African Phthorimaea operculella granulovirus (PhopGV-SA) for the control of the Potato Tuber Moth, Phthorimaea operculella (Zeller) A thesis submitted in fulfilment of the requirements for the degree of MASTER OF SCIENCE At RHODES UNIVERSITY By MICHAEL DAVID JUKES February 2015 i Abstract The potato tuber moth, Phthorimaea operculella (Zeller), is a major pest of potato crops worldwide causing significant damage to both field and stored tubers. The current control method in South Africa involves chemical insecticides, however, there is growing concern on the health and environmental risks of their use. The development of novel biopesticide based control methods may offer a potential solution for the future of insecticides. In this study a baculovirus was successfully isolated from a laboratory population of P. operculella. Transmission electron micrographs revealed granulovirus-like particles. DNA was extracted from recovered occlusion bodies and used for the PCR amplification of the lef-8, lef- 9, granulin and egt genes. Sequence data was obtained and submitted to BLAST identifying the virus as a South African isolate of Phthorimaea operculella granulovirus (PhopGV-SA). Phylogenetic analysis of the lef-8, lef-9 and granulin amino acid sequences grouped the South African isolate with PhopGV-1346. Comparison of egt sequence data identified PhopGV-SA as a type II egt gene. A phylogenetic analysis of egt amino acid sequences grouped all type II genes, including PhopGV-SA, into a separate clade from types I, III, IV and V. These findings suggest that type II may represent the prototype structure for this gene with the evolution of types I, III and IV a result of large internal deletion events and subsequent divergence. PhopGV-SA was also shown to be genetically more similar to South American isolates (i.e. PhopGV-CHI or PhopGV-INDO) than it is to other African isolates, suggesting that the South African isolate originated from South America. Restriction endonuclease profiles of PhopGV-SA were similar to those of PhopGV-1346 and PhopGV-JLZ9f for the enzymes BamHI, HindIII, NruI and NdeI. A preliminary full genome sequence for PhopGV-SA was determined and compared to PhopGV-136 with some gene variation observed (i.e. odv-e66 and vp91/p95). The biological activity of PhopGV-SA against ii 8 -1 P. operculella neonate larvae was evaluated with an estimated LC50 of 1.87×10 OBs.ml being determined. This study therefore reports the characterisation of a novel South African PhopGV isolate which could potentially be developed into a biopesticide for the control of P. operculella. iii Table of contents Abstract ..................................................................................................................................... ii Table of contents ..................................................................................................................... iv List of Figures ....................................................................................................................... viii List of Tables ............................................................................................................................ x List of Equations .................................................................................................................... xii List of Abbreviations ........................................................................................................... xiii Research Outputs .................................................................................................................. xvi Acknowledgements .............................................................................................................. xvii Chapter 1 .................................................................................................................................. 1 Literature review: The potato tuber moth and the baculoviruses ...................................... 1 1.1. Introduction ................................................................................................................... 1 1.1.1. Pests in the South African potato industry ...................................................... 1 1.2. Phthorimaea operculella................................................................................................ 2 1.2.1. Distribution and host range .............................................................................. 3 1.2.2. Life cycle of Phthorimaea operculella ............................................................... 4 1.2.3. Agricultural control options.............................................................................. 5 1.3. Insect viruses ................................................................................................................. 7 1.4. The baculoviruses.......................................................................................................... 8 1.4.1. Taxonomy and nomenclature ........................................................................... 8 1.4.2. Morphological characteristics ........................................................................... 9 1.4.3. Genetic characteristics..................................................................................... 10 1.4.3.1. Transcription genes ............................................................................... 11 1.4.3.2. Replication genes .................................................................................... 11 1.4.3.3. Structural genes ..................................................................................... 12 1.4.3.4. Genes affecting the insect host .............................................................. 12 1.4.4. Life cycle and transmission ............................................................................. 12 iv 1.4.5. Applications of Baculoviruses ......................................................................... 14 1.4.5.1. Expression vectors ................................................................................. 14 1.4.5.2. Biopesticides ........................................................................................... 15 1.4.6. Phthorimaea operculella granulovirus............................................................ 17 1.5. Justification for study ................................................................................................. 18 1.6. Chapter outline: aims and objectives ........................................................................ 18 Chapter 2 ................................................................................................................................ 20 Isolation and morphological identification of a baculovirus isolated from a laboratory colony of Phthorimaea operculella ........................................................................................ 20 2.1. Introduction ................................................................................................................. 20 2.2. Material and Methods ................................................................................................ 22 2.2.1. Rearing of Phthorimaea operculella ............................................................... 22 2.2.2. Occlusion Body Isolation and Purification .................................................... 26 2.2.3. Morphological Identification .......................................................................... 26 2.2.4. Occlusion body enumeration .......................................................................... 27 2.2.4.1. OB enumeration by spectrophotometry .............................................. 27 2.2.4.2. OB enumeration by microscopy ........................................................... 27 2.2.4.3. OB concentration per mg of larval cadaver ........................................ 28 2.3. Results .......................................................................................................................... 28 2.3.1. Insect rearing .................................................................................................... 28 2.3.2. Occlusion body purification ............................................................................ 29 2.3.3. Morphological identification ........................................................................... 30 2.3.4. Occlusion body enumeration .......................................................................... 31 2.4. Discussion..................................................................................................................... 32 Chapter 3 ................................................................................................................................ 35 The genetic characterisation of a novel granulovirus recovered from Phthorimaea operculella larvae ................................................................................................................... 35 3.1. Introduction ................................................................................................................. 35 3.2. Material and Methods ................................................................................................ 37 v 3.2.1. DNA extraction................................................................................................. 37 3.2.2. PCR amplification of granulin, egt, lef-8 and lef-9 genes. ............................ 38 3.2.3.
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