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Browsing by Author "Ibaba, Jacques Davy."

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    Characterization of potato virus Y (PVY) isolates infecting solanaceous vegetables in KwaZulu-Natal (KZN), Republic of South Africa (RSA)
    (2009) Ibaba, Jacques Davy.; Gubba, Augustine.
    Potato virus Y (PVY) is an economically important virus worldwide. In South Africa, PVY has been shown to be a major limiting factor in the production of important solanaceous crops, including potato (Solanum tuberosum L.), pepper (Capsicum annuum L.), tomato (Lycopersicon esculentum Mill.) and tobacco (Nicotiana spp). The variability that PVY displays, wherever the virus occurs, merits the study of the isolates occurring in KwaZulu-Natal (KZN) in the Republic of South Africa (RSA). This characterization will provide a clear understanding of strains/isolates from local vegetables and how they relate to the other PVY strains already identified, as well as information that can be used to manage the diseases they cause. Hence, the aim of this project was to study the biological and genetic properties of PVY isolates infecting potato, tomato and pepper in KZN. Enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies and reverse transcription polymerase chain reaction (RT-PCR) using primers specific to all PVY strains were used to detect the virus in plant material showing PVY-like symptoms collected from various locations in KZN. A total of 39 isolates (18 isolates infecting tomato, 12 infecting potato and 9 infecting pepper) were further differentiated into strains by means of ELISA using strain specific antibodies and RT-PCR using primers specific to the different strains of PVY identified around the world. All PVY isolates infecting tomato and pepper tested positive for the ordinary PVYO strain with both ELISA and RT-PCR. PVY isolates infecting potato were more diverse and comprised the PVYN, PVYNTN and PVYNWilga strains, with mixed infections noted in some cases. The biological properties were studied by mechanically inoculating Chenopodium quinoa, Nicotiana tabacum cv Xanthi, N. tabacum cv Samsun, N. glutinosa, and N. rustica with leaf extracts from plants infected with the different PVY strains detected in this study. All inoculated C. quinoa plants did not show symptoms. All tobacco plants showing symptoms were tested for the presence of PVY by means of ELISA using monoclonal antibodies targeting all strains and electron microscopy using the leaf dip technique. Not all the inoculated tobacco tested positive with ELISA. The symptoms observed were therefore divided into PVY-related and PVY non- related. PVY-related symptoms included vein clearing, mosaic chlorosis, stunting, and vein necrosis. PVY non-related symptoms included wrinkles and leaf distortions. Potyvirus-like particles of about 700 nm were observed under the transmission electron microscope (TEM) from plants showing PVY-related symptoms while rod shaped viral particles of sizes varying between 70 and 400 nm were observed from plants showing non-PVY related symptoms. A portion of the virus genome (1067 bp) covering part of the coat protein gene and the 3’ non-translated region (NTR) of three PVYO isolates infecting tomato, one PVYO isolate infecting pepper and one PVYNWilga isolate infecting potato were amplified, cloned and sequenced. The 5’ NTR, P1, HC-Pro and part of P3 regions (2559 bp) of a PVYN isolate infecting potato were also amplified, cloned and sequenced. Sequence data was compared with selected PVY sequences from different geographical locations around the world. These were available on the NCBI website and subsequently used for phylogenic analyses. The sequenced genomic regions of the PVYN isolate were found to be 99% similar to the New Zealand PVYN isolate (GenBank accession number: AM268435), the Swiss PVYN isolate CH605 (X97895) and the American PVYN isolate Mont (AY884983). Moreover, the deduced amino acid sequence comparison of the genomic regions of the PVYN isolate revealed the presence of five distinct amino acids residues. The three amino acid residues (D205, K400, and E419), which determine the vein necrosis phenotype in tobacco, were also identified. The coat protein and 3’ NTR sequences of all KZN PVYO isolates infecting pepper and tomato were closely similar to each other than to KZN PVYNWilga isolate infecting potato. The phylogenic analysis clustered the KZN PVYN isolate with the European sublineage N, PVYNWilga isolate infecting potato with the American PVYO isolate Oz (EF026074) in the O lineage and all PVYO isolates infecting tomato and pepper in a new sublineage within the O lineage. Taken together, these results point to the presence of PVY in solanaceous vegetables cultivated in KZN and they lay the foundation for the formulation of effective control measure against PVY diseases in KZN.
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    Identification and characterization of viruses infecting cucurbits in the province of KwaZulu-Natal, Republic of South Africa, with the purpose of developing transgenic virus-resistant cucurbits.
    (2016) Ibaba, Jacques Davy.; Gubba, Augustine.; Laing, Mark Delmege.
    The continued presence of cucurbit-infecting viruses across the Province of KwaZulu- Natal (KZN), South Africa requires exploration of alternative methods of controlling these viruses. The aim of this research project was to identify and characterize the viruses infecting cucurbits in KZN with the intention of subsequently developing transgenic cucurbits with broad virus resistance. A systematic virus survey was therefore carried out in all cucurbit growing areas of KZN during the 2011, 2012 and 2013 growing seasons. Symptomatic leaves suspected to be of viral aetiology were sampled and tested using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and reverse transcription-polymerase chain reaction (RTPCR), in order to detect the common viruses causing mosaic and yellowing diseases. Samples were tested for the following viruses: Cucumber mosaic virus (CMV), Squash mosaic virus (SqMV), Moroccan watermelon mosaic virus (MWMV), Watermelon mosaic virus (WMV), Zucchini yellow mosaic virus (ZYMV), Cucurbit yellow stunting disorder virus (CABYV), Beet pseudo-yellows virus (BPYV), Cucumber yellow stunting disorder virus (CYSDV), Lettuce infectious yellows virus (LIYV), and members of the genera Polerovirus and Carlavirus. CMV, BPYV, ZYMV, and MWMV were detected, along with Pepo aphid-borne yellows virus (PABYV), a putative new species in the genus Polerovirus that has been reported in West Africa. MWMV was the most prevalent mosaic-inducing virus and PABYV was the most prevalent yellowing-inducing virus. One common virus symptom consisted of shoe strings leaves and deformed fruits on baby marrows plants (Cucurbita pepo L.). However, the samples tested negative for all the viruses selected. These symptoms were further investigated using Potyvirus universal primers. This led to the detection of a tentative species in the Papaya ringspot virus (PRSV) cluster in the genus Potyvirus. The tentative potyvirus was named Zucchini shoestring virus (ZSSV). Next Generation Sequencing was later used in combination with Sanger Sequencing to elucidate the full genome sequences of MWMV, PABYV, and ZSSV. MWMV isolates from RSA were found to be more closely related to each other than to the isolate from Tunisia. Both PABYV and ZSSV were found to be distinct species in the genera Polerovirus and Potyvirus, respectively, on the basis of their genome organization and the species criteria for each genus. Baby marrow was identified as the most susceptible cucurbit to viral infections in KZN. It was therefore decided to develop baby marrow plants with resistance to the potyviruses identified in the survey, using antisense post-transcriptional gene silencing. A portion of the 5’ coding sequence of the coat protein genes of MWMV, ZYMV and ZSSV were amplified by RT-PCR and inserted into a plant expression vector pEPJ86-m/2N. The expression cassette on Page iii of 136 pEPJ86-m/2N was subsequently sub-cloned into the plant transformation vector pGA482G before being introduced into Rhizobium radiobacter, formerly Agrobacterium tumefaciens, strain LBA4404 (pAL4404)(pBI121) by electroporation. Rhizobium-mediated transformation on baby marrow cotyledon explants was subsequently performed. The resultant putative transgenic regenerated baby marrows plants were subjected to different tests that included PCR to confirm transgene insertion, mechanical inoculation of each virus and DAS-ELISA to evaluate virus resistance. A total of 94 baby marrow plants were successfully regenerated from 250 explants. Out of the 94 plants, 84 were found to have the transgene based on the PCR results. Of these 84 lines, 76 showed resistance to the selected three viruses. Our preliminary results show the potential of using transgenic cucurbits with resistance to three potyviruses as an effective strategy to control virus diseases on cucurbits.