CHAPTER 6: CHARACTERIZATION OF THREE FULL IRIS YELLOW SPOT
6.4 Discussion
128
A B
Figure 6.2 Molecular phylogenetic analyses of the NSs (A) and NSm (B) genes by Maximum Likelihood method based on the Tamura 3-parameter model.
129 viruses that were not meant for study are detected (Martin and Wang, 2011). In this study, Garlic common latent virus, Garlic virus B, Garlic virus C and Shallot virus X were detected (data not shown). Both reference-based sequencing and de novo assembly methods were used in this study for comparison purposes, and there were no significant differences in the IYSV genome recovered.
The fact that viruses other than IYSV were detected in samples sent for NGS shows that mixed and multiple infections are a common occurrence in nature. This compromises the reliance on symptomatology for IYSD diagnosis as symptoms expressed are a result of more than one pathogen.
A major advantage of NGS over other conventional specific approaches like ELISA and PCR is that the latter approaches require reagents designed exclusively to detect their viral target and any variation in the virus genome may cause the assay to fail. NGS is non-targeted and requires no prior knowledge of the target. Therefore, NGS is able to detect existing strains, new variants and even new strains (Adams et al., 2009).
The genomic organization of the Gar-Zim isolate is typical of tospoviruses (Pappu et al., 2009), with the S, M and L RNA segments recovered by both referenced-based mapping and de novo assembly. Though full IYSV genomes were not recovered in this study, NGS enabled the simultaneous recovery of the two full genes on the S RNA segment and the NSm gene on the M segment. With Sanger sequencing, it is normally a time-consuming and expensive process to get so many gene sequences. This study lays the foundation for future studies on the full genome of IYSV in Zimbabwe as only few nucleotides were missing from the recovered segments (data not shown).
IYSV, as already noted, is an important emerging pathogen of alliaceous crops worldwide. Despite its global importance, only a few full genome sequences have been characterized (Gawande et al., 2015). The majority of IYSV disease reports are based on partial N gene sequences. This greatly compromises studies to understand pathogen evolution and management. This is the first study in Africa where more than one full IYSV genes of the same isolate have been described. To date, only two other full IYSV N gene sequences (Accessions KT225547 and KC161369) from Africa have been deposited in public databases.
130 Phylogenetic analysis of the N gene showed no clustering patterns based on geographical location.
This could suggest the possibilities of long-distance migration, recombination and reassortment events in IYSV. Such events are highly prevalent in tospoviruses (Zhang et al., 2016; Margaria et al., 2015). Since IYSV is a pathogen of some ornamental plants that are traded on the world market (CABI, 2016; Bag et al., 2015), it is possible that both the pathogen and its thrips vectors have been unintentionally distributed worldwide. Also, smuggling of live host plants across borders could also have contributed to pathogen’s worldwide distribution. The S RNA segment is known to be substantially more prone to recombination than the M and L RNA segments (Gawande et al., 2015). For either recombination or reassortment to be verified in the Gar-Zim isolate, full genomes of the IYSV segments must be recovered and analyzed.
In conclusion, the characterization of the three IYSV genes from the Gar-Zim isolate lays the foundation for future studies on the full genome of this important pathogen. Knowledge of the full genome is critical in developing management strategies and understanding the evolutionary patterns of IYSV.
References
Adams, I.P., R.H. Glover, W.A. Monger, R. Mumford, E. Jackeviciene, M. Navalinskiene et al.
2009. Next-generation sequencing and metagenomics analysis: a universal diagnostic tool in plant virology. Molecular Plant Pathology 10(4): 537-545.
Bag S., KL Druffel and HR Pappu. 2010. Structure and genome organization of the large RNA of Iris yellow spot virus (genus Tospovirus, family Bunyaviridae). Archives of Virology 155:
275-279.
Bag, S., H.F. Schwartz, CS Cramer, MJ Harvey and HR Pappu. 2015. Iris yellow spot virus (Tospovirus: Bunyaviridae): from obscurity to research priority. Molecular Plant Pathology 16(3): 224-237.
Bag, S., H.F. Schwartz and H.R. Pappu. 2012. Identification and characterization of biologically
131 distinct isolates of Iris yellow spot virus (genus Tospovirus, family Bunyaviridae), a serious pathogen of onion. European Journal of Plant Pathology 134: 97-104.
Bandla, M.D., L.R. Campbell, D.E. Ullman and J.L. Sherwood. 1998. Interaction of Tomato spotted wilt tospovirus (TSWV) glycoproteins with a thrips midgut protein, a potential cellular receptor for TSWV. Phytopathology 88: 98-104.
Bankevich, A., S. Nurk, D. Antipov, A.A. Gurevich, M. Dvorkin, A.S. Kulikov et al. 2012.
SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing.
Journal of Computational Biology 19: 455-477.
Bolger, A.M., M. Lohse and B. Usadel. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15): 2114-2120.
Bushnell, B. 2014. BBMap short read aligner. https://sourceforge.net/projects/bbmap). Accessed on June 11, 2017.
CABI. 2016. Iris yellow spot virus datasheet 28848. (www.cabi.org/isc/datasheet/28848).
Accessed on July 7, 2016.
Cortês, I., I.C. Livieratos, A. Derks and R Kormelink. 1998. Molecular and serological
characterization of Iris yellow spot virus, a new and distinct Tospovirus species.
Phytopathology 88: 1276-1282.
Gasteiger, E., C. Hoogland, A. Gattiker, S. Duvaud, M.R. Wilkins, R.D. Appel and A. Bairoch.
2005. Protein identification and analysis tools on the ExPASy Server. In: The Proteomics Protocols Handbook. J.M. Walker (eds). Humana Press, USA.
Gawande, S.J., V.S. Gurav, D.P. Martin, R. Asokan and J. Gopal. 2015. Sequence analysis of Indian Iris yellow spot virus ambisense genomic segments: evidence of interspecies RNA recombination. Archives of Virology 160: 1285-1289.
http://www.bioinformatics.babraham.ac.uk/projects/fastqc. Accessed on 10 June 2017.
Karavina, C and A. Gubba. 2017. Iris yellow spot virus in Zimbabwe: Incidence, severity and
132 characterization of Allium-infecting isolates. Crop Protection 94: 69-76.
Karavina C., J.D. Ibaba and A. Gubba. 2016. First report of Iris yellow spot virus infecting onion in Zimbabwe. Plant Disease 100(1): 235.
King, A.M.Q., M.J. Adams, E.B. Carstens and E.J. Lefkowitz (eds). 2012. Virus taxonomy- classification and nomenclature of viruses. Ninth Report of the International Committee on Taxonomy of Viruses. Elservier Academic Press, Amsterdam, The Netherlands, pp725- 741.
Kreuze J. F., A. Perez, M. Untiveros, D. Quispe, S. Fuentes, I. Barker and R. Simon. 2009.
Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a genetic method for diagnosis discovery and sequencing of viruses. Virology 388: 1-7.
Margaria, P., L. Miozzi, M. Ciuffo, H. Pappu and M. Turina. 2015. The first complete genome sequences of two distinct European Tomato spotted wilt virus isolates. Archives of Virology 160: 591-595.
Martin, J.A. and Z. Wong. 2011. Next-generation transcriptome assembly. Nature Reviews Genetics 12: 671-682.
Pappu, H. R., R. A. C. Jones and R. K. Jain. 2009. Global status of Tospovirus epidemics in diverse cropping systems: Successes achieved and challenges ahead. Virus Research 141: 219-236.
Srinivasan R, S. Sundaraj, H.R. Pappu, S. Diffie and D.G. Riley. 2012. Transmission of Iris yellow spot virus by Frankliniella occidentalis and Thrips tabaci (Thysanoptera: Thripidae).
Journal of Economic Entomology 105: 40-47.
Tamura, K., G. Stecher, D. Peterson, A. Filipski and S. Kumar. 2013. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725- 2729.
Walsh K., J. North, I Barker and N. Boonham. 2001. Detection of different strains of Potato virus
133 Y and their mixed infections using competitive fluorescent RT-PCR. Journal of Virological Methods 91: 167-173.
www.imed.ucm.es/Tools/sias.html. Accessed on June 13, 2017
Zhang, Z., D. Wang, C. Yu, Z. Wang, J. Dong, K. Shi and X. Yuan. 2016. Identification of three new isolates of Tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses. Virology Journal 13:8 doi 10.1186/s12985-015-0457-3.
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