Alternative hosts and seed transmissibility of Soybean blotchy mosaic virus

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Preface

Glycine max (Fabaceae, Phaseoleae) is a nutritious, annual leguminous plant species native to East Asia. Soybean, or Greater bean as it is sometimes known, was used as a source of protein and oil by the Chinese as early as 2500 B.C (Hartman et al. 1999). Soybean was only introduced into the western world in the 19th century, and today sustainable and industrious soybean production is of global importance due to its use for both human consumption and animal feed. In 2016, 742 000 tonnes of soybean was produced on approximately 502 800 hectares in South Africa (faostat.org). However, a number of viral, bacterial, fungal and insect pests and pathogens affect the production of soybean each year. Implementation of control measures aimed at limiting these production losses relies on a clear understanding of the pests and pathogens affecting soybean.

Taxonomy of the family Rhabdoviridae

Prior to 2016, rhabdoviruses were classified in the order Mononegavirales by the International Committee on Taxonomy of Viruses (ICTV) based on their non-segmented, linear, single-stranded, negative sense RNA genomes and lipoprotein membrane (envelope) (Tordo et al. 2005; Khan and Dijkstra 2006; Dietzgen and Kuzmin 2012). The Mononegavirales, established in 1991, initially accommodated the families Rhabdoviridae, Filoviridae and Paramyxoviridae, but to date, eight families have been recognized (Rhabdoviridae, Filoviridae, Paramyxoviridae, Bornaviridae, Nyamiviridae, Mymonaviridae, Pneumoviridae and Suniviridae) (Amarasinghe et al. 2017). Members are classified in the respective families based on the shape of viral particles, genome complement and organization and mechanisms of gene expression (Jackson et al. 2005; Khan and Dijkstra 2006).

Morphology and particle structure

With the exception of the dichorha- and varicosaviruses which lack a lipoprotein membrane, members of the family Rhabdoviridae have large enveloped particles with a characteristic bullet shaped (animal and human rhabdoviruses) or bacilliform (plant rhabdoviruses) morphology (Figure 1.2A), from which the family derived its name, as rhabdos is a Greek word meaning rod shaped (Khan and Dijkstra 2006; Jackson et al. 2005; Kormelink et al. 2011; Dietzgen and Kuzmin 2012; Amarasinghe et al. 2017). Particles vary in length and width, ranging from 100-430 nm to 45-100 nm respectively (Dietzgen and Kuzmin 2012). Due to the distinctive nature of rhabdovirus particle morphology, infection of plant material by members of the family is often confirmed by electron microscopy of plant extracts or of thin sections of plant material, as viral particles are easily distinguished from cell components (Jackson et al. 2005). Many members of the family have been identified and described only in this manner, and thus require additional molecular characterization to confirm their inclusion in the group (Jackson et al. 2005).

Plant nucleo- and cytorhabdoviruses

The plant-adapted rhabdoviruses differ from their human and animal infecting counterparts in two regards. They are able to spread systemically through host plants, which are facilitated by additional movement-associated genes encoded in their genomes, and plant rhabdoviruses are also uniquely adapted to counteracting RNA silencing, an innate immune response of their hosts, plants and insects (Kormelink et al. 2011; Mann et al. 2016). The genera Nucleorhabdovirus and Cytorhabdovirus are distinguished within the family Rhabdoviridae based on the site of replication and accumulation of viral particles in plant cells (Figure 1.3 and Figure 1.4) (Jackson et al. 2005; Dietzgen and Kuzmin 2012). Species are distinguished based on host range and their vectors, and more recently whole genome sequences (Dietzgen and Kuzmin 2012). Crops and weeds in tropical, sub-tropical and temperate regions are infected, with disease symptoms ranging from stunting, clearing and yellowing of veins to mosaic or mottling and necrosis (Jackson et al. 2005).

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Acknowledgements
Abstract
Chapter 1 Genomics, biology and vector relationships of plant nucleo- and cytorhabdoviruses
1.1 Taxonomy of the family Rhabdoviridae
1.2 Morphology and particle structure
1.3 Plant nucleo- and cytorhabdoviruses
1.3.1 Genus Nucleorhabdovirus
1.3.2 Genus Cytorhabdovirus
1.3.3 Soybean blotchy mosaic virus
1.4 Genome organization
1.5 Phylogenetic relationships and genetic diversity
1.6 Ecological and epidemiological factors affecting the spread of plant nucleo- and cytorhabdoviruses
1.6.1 Insect vectors of plant nucleo- and cytorhabdoviruses
1.6.2 Modes of transmission
1.6.2.1 Persistent, propagative transmission of plant rhabdoviruses
1.6.2.2 Horizontal and vertical transmission of plant nucleo- and cytorhabdoviruses in insect vectors
1.6.2.3 Peragallia caboverdensis, a leafhopper vector of SbBMV
1.6.3 The role of alternative plant hosts
1.7 Seed, pollen and mechanical transmission
1.8 Conclusion
1.9 Literature cited
Chapter 2 Alternative hosts and seed transmissibility of Soybean blotchy mosaic virus
2.1 Abstract
2.2 Acknowledgements
2.3 Literature cited
Chapter 3 Development of a strand-specific RT-PCR to detect the positive sense replicative strand of Soybean blotchy mosaic virus
3.1 Abstract
3.2 Introduction
3.3 Materials and Methods
3.3.1 Primer design
3.3.2 RNA extraction
3.3.3 Reverse transcription
3.3.3.1 Reverse transcription of the genomic (negative) strand of SbBMV
3.3.3.2 Reverse transcription of the antigenomic (positive) strand of SbBMV
3.3.3.3 Reverse transcription of falsely primed cDNAs
3.3.3.4 Reverse transcription of internal control gene
3.3.4 PCR amplification and Sanger sequencing
3.3.4.1 Diagnostic Soyblotch RT-PCR
3.3.4.2 Soyblotch pss-RT-PCR
3.3.4.3 PCR amplification of RuBisCo gene region
3.3.5 Sensitivity of Soyblotch pss-RT-PCR
3.4 Results
3.4.1 Specificity of Soyblotch pss-RT-PCR
3.4.1.1 Absence of detection of genomic RNA and misprimed cDNAs using the Soyblotch pss-RT-PCR
3.4.1.2 Specificity of Soyblotch pss-RT-PCR for positive sense strand
3.4.2 Sensitivity of Soyblotch pss-RT-PCR
3.4.3 Screening of SbBMV isolates using Soyblotch pss-RT-PCR
3.5 Discussion
3.6 Acknowledgements
3.7 Literature cited
Chapter 4 Transmission of Soybean blotchy mosaic virus by the leafhopper, Peragallia caboverdensis Lindberg (Hemiptera: Cicadellidae: Megophthalminae)
4.1 Abstract
4.2 Introduction
4.3 Materials and Methods
4.3.1 Survey for leafhoppers in a soybean production area with a high SbBMV incidence
4.3.2 Identification of leafhoppers based on morphology
4.3.3 Non-destructive DNA extraction from voucher specimens
4.3.4 RNA extraction from leafhoppers
4.3.5 Detection of SbBMV in Pcaboverdensis using RT-PCR
4.3.6 RT-PCR amplification and Sanger sequencing of COI and H3 in P caboverdensis
4.3.7 Phylogenetic analysis
4.3.8 Statistical analysis
4.4 Results
4.4.1 Leafhoppers identified during survey
4.4.2 Detection of SbBMV in Pcaboverdensis
4.4.3 Phylogenetic analysis and barcoding of Pcaboverdensis
4.5 Discussion
4.6 Acknowledgements
4.7 Literature cited
Chapter 5 Diversity of partial RNA-dependent RNA polymerase gene sequences of Soybean blotchy mosaic virus isolates from different host-, geographical- and temporal origins
5.1 Abstract
5.2 Acknowledgements
5.3 Literature cited
Chapter 6 General Conclusion
6.1 General discussion and future prospects
6.2 Literature cited

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Soybean blotchy mosaic virus: Molecular characterization and seasonal persistence

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