Control in the field relies heavily on chemical insecticides which, although effective, have negative environmental implications and are associated with the development of pesticide resistance. Alternative methods of control are urgently required and there is increasing interest worldwide in the use of entomopathogenic microbes as biocontrol agents, specifically a group of naturally occurring double-stranded DNA viruses belonging to the Baculoviridae. Baculoviruses are divided into four genera including Alphabaculovirus (lepidopteran-specific nucleopolyhedrovirus), Betabaculovirus (lepidopteran-specific granulovirus), Gammabaculovirus (hymenopteran-specific nucleopolyhedrovirus) and Deltabaculovirus (dipteran-specific nucleopolyhedrovirus). Baculoviruses are characterised by a bi-phasic lifecycle in which at least two different virion phenotypes are produced. Following infection of the larval mid-gut cells, budded virus is formed which initiates a systemic infection within the infected insect. Secondary infection results in the production of occluded virus which becomes enclosed in a protein coat of either granulin (in the case of granuloviruses) or polyhedron (in the case of nucleopolyhedroviruses). These occlusion bodies (Figure 2, image source: Abdulkadir et al., 2013) are responsible for horizontal transmission of the virus from host to host and serve to protect the virions from damage in the environment during the non-infectious stage of the lifecycle. Several features of baculoviruses including host-specificity, a favourable biosafety profile and pathogenicity against the larval stages of the host (Figure 3) have led to their increased application as biocontrol agents against a variety of agricultural pests.

Baculovirus biopesticides are used in South Africa as part of integrated pest management programmes which aim to reduce resistance to insecticides and unwanted survival of the pests. Examples of locally manufactured products include Cryptogran® (River Bioscience Pty. Ltd), formulated with the T. leucotreta granulovirus (CrleGV), and HelicovirTM (River Bioscience Pty. Ltd), containing the H. armigera nucleopolyhedrovirus (HearNPV), and are applied in the field for the control of false codling moth and African bollworm respectively. In addition, Madex® (Andermatt, Switzerland) and CarpovirusineTM (Arysta, France) both formulated with a Mexican strain of the C. pomonella granulovirus, CpGV-M, are imported for the control of codling moth. CrleGV and HearNPV products produced by Andermatt, such as Cryptex and Bolldex, are also imported. As with chemical insecticides, resistance development in insect populations to repeated application of viral formulations complicates their effectiveness as has been observed in codling moth, where field populations in Europe developed resistance to CpGV-M. Therefore, the recovery and characterisation of genetically different virus isolates with greater virulence is vital to complement existing baculovirus insecticides in South Africa, and also to manage resistance should it occur in the field.

One project conducted within the Biological control Research Group at Rhodes University involves bioprospecting for novel South African virus isolates either in the field or in laboratory reared insect populations, with a view to their development and application as baculovirus biopesticides against a variety of economically important insect pests in the field. Occlusion bodies of novel isolates are recovered from diseased insect larvae by glycerol gradient purification and undergo morphological characterisation using transmission electron microscopy.

Thereafter, viruses are genetically characterised using a variety of techniques including whole genome sequencing and analysis, PCR amplification and sequencing of selected core genes and also restriction endonuclease analysis of genomic DNA extracted from gradient-purified occlusion bodies (Figure 4). Sequences and DNA profiles are then compared to those of reference strains whose genomes are available on GenBank.

To characterise novel viruses in terms of biological activity (virulence) against insect hosts, droplet or surface dose bioassays are performed using different concentrations of purified occlusion bodies fed to neonate larvae (Figure 5). Lethal concentration and

lethal time (LC50 and LT50 respectively) are evaluated, providing an indication of virulence and speed of kill for the virus being tested.

This research is a collaboration between the departments of Biochemistry and Microbiology and Zoology and Entomology at Rhodes University and Citrus Research International. Recent highlights in our research include not only the recovery of five novel T. leucotreta granuloviruses from laboratory reared insect colonies, but also the isolation and characterisation of novel South African strains of viruses targeting the diamondback moth, the potato tuber moth, the litchi moth, the codling moth and the African bollworm (Figure 6).













Authors:
Caroline Knox (PhD); Michael Jukes (MSc)
Department of Biochemistry and Microbiology, Rhodes University, Grahamstown








Professor Martin Hill
Department of Zoology and Entomology, Rhodes University, Grahamstown









Sean Moore (PhD)
Citrus Research International, Port Elizabeth









Further reading:

1. Abdulkadir, F., Knox, C., Marsberg, T., Hill, M.P. and Moore, S.D. 2015. Genetic and biological characterisation of a novel Plutella xylostella granulovirus, PlxyGV-SA. Biocontrol. 60(4): 507–515.
2. Knox, C., Moore, S.D., Luke, G.A. and Hill, M.P. 2015. Baculovirus-based strategies for the management of insect pests: a focus on development and application in South Africa. Biocontrol Science and Technology. 25(1): 1-20.
3. Opoku-Debrah, J., Hill, M., Knox, C.M. and Moore, S.D. 2014. Comparison of the biology of geographically distinct populations of the citrus pest, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) in South Africa. African Entomology. 22(3): 530–537.
4. Jukes, M.D., Knox, C.M., Hill, M.P. and Moore, S.D. 2014. The isolation and genetic characterisation of a South African strain of Phthorimaea operculella granulovirus, PhopGV-SA. Virus Research 183: 85-88.
5. Opoku-Debrah, J., Hill, M., Moore, S. and Knox, C. 2013. Overcrowding of false codling moth, Thaumatotibia leucotreta (Meyrick) leads to the isolation of five new Cryptophlebia leucotreta granulovirus (CrleGV-SA) isolates. Journal of Invertebrate Pathology 112: 219–228.
6. Abdulkadir, F., Marsberg, T., Knox, C., Hill, M.P., Moore, S.D. 2013. Morphological and genetic characterization of a South African Plutella xylostella granulovirus (PlxyGV) isolate. African Entomology 21(1): 168-171.
7. Moore, S.D., Kirkman, W., Richards, G.I. & Stephen, P. 2015. The Cryptophlebia leucotreta granulovirus – 10 years of commercial field use. Viruses 2015, 7 : 1284-1312; doi:10.3390/v7031284.
8. Moore, S. D., Pittaway, T., Bouwer, G., & Fourie, J. G. 2004. Evaluation of Helicoverpa armigera nucleopolyhedrovirus, HearNPV, for control of Helicoverpa armigera Hübner (Noctuidae: Lepidoptera) on citrus in South Africa. Biocontrol Science and Technology. 14: 239–250.
9. Moore, S. D., Richards, G. I., Chambers, C., & Hendry, D. 2014. An improved larval diet for commercial mass rearing of the false codling moth, Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). African Entomology. 22: 216–219.