For example, no study on tospovirus has been undertaken in Zimbabwe in the last two decades. While most of the new tospovirus species have been reported in the last two and a half decades, it is not known if any of them occur in Zimbabwe.
LITERATURE REVIEW
Origin and distribution of tospoviruses
TSWV and INSV, with a wide range of decorative host plants (Sether and DeAngelis, 1992; EPPO, 2004), have been detected at international ports of entry. Species of thrips, some of which transmit these tospoviruses, have also been found hitchhiking on ornamental plants moving in international trade.
Taxonomy
Characteristics of tospoviruses
- Morphological and physicochemical properties
- Genomic organization
- Host range
A few copies of the virion-associated RNA-dependent RNA polymerase (RdRp) are shown (purple) in association with the RNPs (Whitfield et al., 2005). More than 1300 plant species from at least 92 families of monocots and dicots are hosts of tospoviruses (Parrella et al., 2003).
Tospoviral replication
Transmission
Similarly, plant species that do not support thrips development are “dead ends” for the viruses, regardless of the number of infected thrips species feeding on them (Whitfield et al., 2005). This time allows the virus to move and multiply in the insect gut and salivary glands (Ullman et al., 2005).
Effects of tospovirus infection
- Cytopathological effects
- Symptom expression
Virus acquisition occurs only during the larval stages, after which the virus is transstadially transmitted to the adult. These symptoms vary with host species, cultivar, virus strain and/or isolate, plant age, season, and environment (German et al., 1992; Sevik and Arli-Sokmen, 2012).
Detection and diagnosis of tospoviruses
- Sampling and field detection
- Diagnosis
For viruses to be visualized, their concentration must be at least 105-6 particles per ml (Biel et al., 2004). Rare variant sequences can be identified (Reis-Filho, 2009) and complete genome sequences are identified in an unbiased manner (Kreuze et al., 2009).
Management of tospovirus diseases
- Biological control
- Chemical control
- Cultural control
- Physical control
- Host plant resistance
In Kenya, Biritia et al. 2014) assessed onion varieties for resistance to IYSV and Trips tabaci. This has resulted in the development of tospovirus-resistant transgenic plants (Gubba et al., 2002; Uhrig, 2007).
Challenges in managing tospoviruses
Resistance-breaking strain of the Tomato Spotted Wilt Virus (TSWV) on resistant pepper varieties in Hungary. Isolation and molecular characterization of isolates of the Tomato Spotted Wilt Virus (TSWV) occurring in South Africa.
KNOWLEDGE AND PERCEPTIONS OF PLANT VIRAL DISEASES BY
- Introduction
- Materials and Methods
- Study area
- Sampling procedure and selection of participants
- Data collection
- Data analysis
- Results
- Socio-economic characteristics of respondents
- Major plant viruses in Zimbabwe
- Training for disease identification
- Respondents’ perceptions of plant viruses and methods used in virus disease identification
- Opinions on improving virus diseases awareness
- Knowledge of TSWV/tospoviruses by respondents
- Discussion
The state employed 77.1% of the technical staff, and only 20.1% in the private sector. 68.9% of farmers and 40.7% of technical staff recommended the use of insecticides to control TSWV/tospoviruses. The use of certified seed and fumigation was suggested by 5.8% of farmers and 14.8% of technical staff.
To improve TSWV/tospovirus awareness, 27.1% of technical staff recommended “college/university/school education” while 19.2% recommended.
DETECTION AND CHARACTERIZATION OF TOMATO SPOTTED WILT
Introduction
It causes losses estimated at 1 billion. USD annually for several important ornamental, food, and cash crops worldwide (Goldbach and Peters 1994; Parrella et al. 2003). The S-RNA encodes the non-structural protein (NSs) in the viral sense and the nucleocapsid (N) protein in the non-viral sense (Whitfield et al. The TSWV virion is quasi-spherical, 80–120 nm in diameter and enclosed by a double membrane (German et al. 1992).
Necrotic spots are also present on peppers, often showing ring patterns (Turina et al. 2012).
Materials and Methods
- Sampling and field detection of tospoviruses
- Double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA)
- Molecular characterization of TSWV
On-site detection of tospovirus was performed using immunostrips (Loewe® Biochemica GmbH, Germany), which were able to simultaneously detect tomato leaf spot orthotospovirus (TSWV), tomato chlorotic spot orthotospovirus (TCSV) and peanut ringspot orthotospovirus (GRSV), in according to the manufacturer's instructions. Total RNA was extracted from leaf samples stored in RNAlater® solution using the Quick-RNATM MiniPrep Kit (Zymo Research, Irvine, USA) according to the manufacturer's instructions. PCR products corresponding to the target genes were purified using the QIAQuick DNA Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.
Cloning was performed using the TOPO-TA Cloning Kit (Invitrogen, USA) according to the manufacturer's instructions.
Results
- Geographical distribution of TSWV in Zimbabwe
- Field detection of tospoviruses using immunostrips
- Serological characterization of TSWV
- Molecular and phylogenetic analyses
The bootstrap consensus tree derived from 1000 replicates is taken as a representation of the evolutionary history of the taxa analyzed. Most TSWV-positive locations were within 30 km of Harare's central business district. None of the samples that had been negative by DAS-ELISA were positive by PCR, even when different primers were used.
When compared among themselves, the partial N gene of the Zimbabwean isolates showed at least 99.02% and 93.99%.
Discussion
Overcoming host- and pathogen-mediated resistance in tomato and tobacco maps to the M RNA of Tomato spotted wilt virus. 83 production and their role as alternative hosts for tomato spotted wilt virus and its vector Frankliniella occidentalis. Marker-assisted selection for linkage resistance to tomato spotted wilt virus and Phytophthora infestans in tomato.
Evaluation of the effect of time of infection with Tomato spotted wilt virus (TSWV) on some yield components of tomato.
IRIS YELLOW SPOT ORTHOTOSPOVIRUS IN ZIMBABWE: INCICDENCE,
Introduction
Iris yellow spot orthotospovirus, IYSV, an emerging pathogen of alliaceous crops, has been reported in many countries worldwide (Bag et al., 2015). Like other tospoviruses, IYSV is transmitted exclusively by thrips (Thysanoptera: Thripidae) in a circulative-propagative mode (Whitfield et al., 2005). Currently, Thrips tabaci Lindeman and Frankliniella fusca (Hinds) are the known IYSV vectors (Srinivasan et al., 2012; Bag et al., 2015).
Recent studies have reported up to 60% of Iris yellow spot disease (IYSD) incidence in alliaceous crops in Zimbabwe (Karavina et al., 2016a, b).
Materials and Methods
- Disease surveys
- Serological diagnosis
- Transmission studies
- Electron microscopy studies
- Molecular detection
- Phylogenetic analysis of the partial IYSV N gene
In the final step, 0.2 ml of the substrate buffer solution containing the dissolved substrate tablet was applied to the microtiter plate. The nucleotide sequences of the IYSV isolates reported in this study have been deposited in GenBank under the accession numbers given in Table 4.2. From the bootstrap consensus tree it was inferred that 1000 replicates were taken to represent the evolutionary history of the taxa analyzed.
Genetic distances between Zimbabwean IYSV isolates and those from the rest of the world were calculated from the Tamura 3-parameter model (Tamura, 1992).
Results
- Geographical distribution, incidence and severity of Iris yellow spot orthotospovirus in
- Iris yellow spot orthotospovirus symptomatology in Zimbabwe
- IYSV natural host range and detection by serological, electron microscopy and transmission
- Molecular detection and Phylogenetic analysis
When total RNAs of ELISA-positive samples were subjected to RT-PCR amplification using primers specific for the IYSV N gene, a 236 bp amplification was observed from infected tissues but not from healthy controls. Zimbabwean IYSV isolates shared identity and similarity at the nucleotide and amino acid levels, respectively. Zimbabwean IYSV isolates clustered with isolates from Europe (Bosnia and Herzegovina, Greece, Italy, Israel, Spain and Slovenia), the Americas (Brazil, Ecuador, Guatemala, Peru and the USA), Canada, Asia (Japan, India, Sri Lanka, Tajikistan and Pakistan), Australia, New Zealand and Africa (Egypt, Kenya, Mauritius and South Africa).
100 Figure 4.3: Phylogenetic relationships of partial N gene sequences of Zimbabwean IYSV isolates and those in GenBank from different regions of the world.
Discussion
Structure and genome organization of the large RNA of Iris yellow spot virus (genus Tospovirus, family Bunyaviridae). Population dynamics of Iris yellow spot virus in onion fields and lisianthus greenhouses in Japan. Phylogenetic analysis of Iris yellow spot virus isolates from onion (Allium cepa) in Georgia (USA) and Peru.
First report of Iris yellow spot virus in onion bulb and seed production fields in the Réunion Islands.
NATURAL NON-ALLIUM HOSTS OF IRIS YELLOW SPOT ORTHOTOSPOVIRUS
- Introduction
- Materials and Methods
- Weed and thrips samples collection
- Serological detection
- Molecular characterization and Phylogenetic analysis
- Results
- Serological diagnosis
- Molecular detection and Phylogenetic analysis
- Discussion
Green foxtail (Setaria viridis), a naturally infected grass host of Iris yellow spot virus in Utah. First report of iris yellow spot virus in onion bulb and seed production fields on Reunion Island. Identification of new alternative weed control agents for Iris yellow spot virus in the Pacific Northwest.
Weed hosts for onion thrips (Thysanoptera: Thripidae) and their potential role in the epidemiology of Iris yellow spot virus in an onion ecosystem.
CHARACTERIZATION OF THREE FULL IRIS YELLOW SPOT
- Introduction
- Materials and Methods
- Sources of materials used
- Serological detection
- RNA extraction and RT-PCR
- Sample preparation, sequencing and data analysis for Next-Generation Sequencing
- IYSV sequence analysis and phylogeny
- Results
- Serology and RT-PCR analyses
- RNA quality assessment and NGS data analysis
- Sequence characteristics and phylogenetic analyses of the N, NSs and NSm genes from the IYSV
- Discussion
The molecular weight (Mw) of the proteins was determined using the online ExPASy bioinformatics tool (Gasteiger et al., 2005). Phylogenetically, the NSs gene of the IYSV Gar-Zim isolate clustered with the homologous sequences of isolates from India and the Netherlands (Figure 6.2A). As for the NSm gene of the Gar-Zim isolate, it also clustered with homologous sequences from IYSV isolates from Brazil (AF213677) and the USA (FJ361359) (Figure 6.2B).
The genomic organization of the Gar-Zim isolate is typical of tospoviruses ( Pappu et al., 2009 ), with S, M, and L RNA segments recovered from both reference-based mapping and de novo assembly.
MARKER-ASSISTED IDENTIFICATION OF TOMATO SPOTTED WILT
Introduction
Sometimes thrips populations are suppressed, but this does not prevent disease spread (Soler et al., 2003). Wild relatives of domesticated tomatoes have been screened for resistance to TSWV isolates (Stevens et al., 1994). CAPS and RFLP markers are based on the presence of SNPs or insertion/deletion (INDEL) mutations (Nasu et al., 2002).
SNPs have also been used to develop SNP markers useful for MAS (Wu et al., 2008).
Materials and Methods
- Genetic material
- DNA extraction
- PCR amplification
- Gel purification, sequencing and analysis
Plants carrying this allele can limit systemic virus spread, showing only localized symptoms represented as small local lesions caused by a hypersensitive response (Dianese et al., 2010). MAS was applied to identify TSWV-resistant tomato germplasm using markers linked to the Sw-5 gene (Shi et al., 2011; Lee et al., 2015). The use of gene-based markers is beneficial for increasing the accuracy of MAS compared to the use of markers generated based on DNA polymorphisms located along the gene of interest (Salgotra et al., 2014).
Consensus sequences were generated from the sequences by alignment with ClustalW in MEGA6 ( Tamura et al., 2013 ).
Results and Discussion
- Evaluation of the tomato genotypes for TSWV resistance using the Sw-5b gene-based and SNP
The accession lines carrying the Sw-5b gene can be advanced for use in breeding for TSWV resistance. The study showed that MAS is a rapid method that can be used to screen tomato genotypes for the Sw-5b gene. Development of a single-nucleotide polymorphism marker for the Sw-5b gene conferring disease resistance to tomato spotted wilt virus in tomato.
Mapping the Sw-5 locus for tomato spotted wilt virus resistance in tomato using RAPD and RFLP analyses.
THESIS OVERVIEW
- Introduction
- Knowledge and percetions of plant viral diseases by different stakeholders in Zimbabwe’s
- Detection and characterization of Tomato spotted wilt orthotospovirus infecting field and
- Iris yellow spot orthotospovirus in Zimbabwe: Incidence, severity and characterization of
- Natural non-Allium hosts of Iris yellow spot orthotospovirus in Zimbabwe
- Characterization of three full Iris yellow spot orthotospovirus genes of a garlic-infecting isolate
- Marker-assisted identification of Tomato spotted wilt orthotospovirus-resistant tomato
- Way forward
The virus was found in only 50% of the districts surveyed, mainly in the country's main agricultural region. Furthermore, a mixed infection of TSWV and PVY in Capsicum annuum was reported for the first time in Zimbabwe. The weed hosts identified in this study are important in IYSV epidemiology because, as summer weeds, they provide a green bridge for virus survival in the off-season Allium species in Zimbabwe.
This needs to be done in Zimbabwe to overcome challenges associated with the movement of pathogens of quarantine importance across international borders.
