The isolation, identification, and characterisation of novel insect viruses for the control of emerging and existing agricultural pests

Abstract

The intensification and expansion of agriculture are among the most prominent global changes driven by human activity. As the human population continues to grow, one of the major global challenges is ensuring adequate food production. Insects play an important role in crop production because of the ecosystem functions that they provide. The ecosystem functions that insects provide are often disrupted in agricultural ecosystems. The modification of habitats by humans, such as selecting crops for higher yield, larger size or those that have higher nutritional value, and the cultivation of monocultures for maximum productivity, all contribute to the emergence of insect pests. Additionally, changing climatic conditions or changes in farming practices may result in changes in the status of certain pests in different agricultural crops. In various studies, viruses that are harboured by several agriculturally important pests in South Africa, including Plutella xylostella, Phthorimaea operculella and Thaumatotibia leucotreta, have been isolated and biologically characterised, and some are now commercially available as biopesticides. In addition, the RNA virus family Dicistroviridae are viruses that infect arthropods. An example of a virus from this family is the Cricket paralysis virus (CrPV), which was first isolated from Australian field crickets. This virus is known to infect both crickets and fruit flies. As such, the use of this virus may find application in the management and control of fruit flies. To safeguard crop production in South Africa, continued bioprospecting for novel biological control options, particularly viruses, against major, minor, and emerging insect pests is important. The aim of this thesis was to isolate, identify, and characterise novel viruses from various insect pests that already impact, or have the potential to impact one or more important crops. Serrodes partita (fruit-piercing moth), Thaumatotibia batrachopa (macadamia nut borer), Ectomyelois ceratoniae (carob moth), and Acheta domesticus (house cricket) were the selected species for analysis in this study, because each species contributes meaningfully to the agricultural ecosystem. Species identification of these species was conducted using molecular genetic techniques. Genomic DNA was extracted from the larval samples of S. partita, T. batrachopa, and E. ceratoniae, and the adults of A. domesticus. Thereafter, PCR amplification of the Cytochrome c oxidase subunit I (COI) gene was evaluated. Sequencing and BLAST analysis confirmed the identity of each species. There have been no reports describing the isolation of viruses from S. partita, E. ceratoniae, and T. batrachopa. Isolating novel baculoviruses from these lepidopteran species holds potential for their use as microbial control agents and for their development as biopesticides. Symptomatic larval cadavers of S. partita, T. batrachopa, and E. ceratoniae were collected, and baculovirus occlusion bodies (OBs) were purified from these samples. Genomic DNA (gDNA) was extracted from the purified OBs and was used for PCR amplification of the polyhedrin/granulin (polh/gran) gene from multiple samples, and the late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes from select samples. Sanger sequencing of the PCR amplicons was conducted, followed by bioinformatic analysis. Sequencing results suggest that a novel baculovirus has been isolated from S. partita larvae. The virus was identified as an Alphabaculovirus, and was basal to a monophyletic clade which includes species such as Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) and Lymantria xylina nucleopolyhedrovirus (LyxyNPV), with Kimura two-parameter distance matrices indicating this virus to be a novel species, hereafter referred to as SepaNPV. Whole genome Illumina sequencing was conducted using the SepaNPV genomic DNA, and a de novo assembly was conducted to reassemble the genome sequence, using the generated Next-generation sequencing (NGS) data. Results indicate a genome size of 129953 bp, with a GC content of 54.9 %, and a preliminary annotated genome with approximately 128 open reading frames (ORFs). Additionally, infection assays were conducted to test the host range of SepaNPV against four potential host species. Data indicates that T. leucotreta, Helicoverpa armigera, Spodoptera frugiperda and Eldana saccharina are not susceptible. Multiple T. batrachopa larvae were also evaluated and shown to be infected with Cryptophlebia leucotreta granulovirus (CrleGV). Furthermore, the final component of the study was to develop a screening method to detect the presence of CrPV in A. domesticus samples. A plasmid-based two-step reverse transcription polymerase chain reaction (RT-PCR) CrPV positive control sample was effectively developed. Additionally, an RNA isolation protocol was established using InvitrogenTM PhasemakerTM Tubes in conjunction with the InvitrogenTM TRIzolTM reagent for RNA isolation and a RT-PCR amplification. The protocol was used for isolating RNA from symptomatic A. domesticus samples; however, CrPV was not detected in the samples. This research provides new insights into baculovirus diversity and host associations by documenting novel virus-host relationships. It expands the known host range of CrleGV and presents the first identification of a baculovirus from S. partita. Through the isolation and characterisation of these viruses, the study enhances our broader understanding of baculovirus biology, host ecology, and insect-virus interactions. Moreover, the development of a screening method provides a valuable tool for future detection of CrPV infection in symptomatic A. domesticus samples.