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.

Abstract

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.