First report of Groundnut ringspot virus (GRSV) infecting tobacco (Nicotiana tabacum L.) in South Africa.

Tobacco (Nicotiana tabacum L.) is an important industrial crop from the Solanaceae family cultivated for its leaves in approximately 100 countries worldwide. In South Africa (SA), tobacco is grown in five of its nine provinces: Limpopo, North West, Western Cape, Eastern Cape, and Mpumalanga. The producer price for unmanufactured tobacco is approximately US$3570/ton annually (FAO, 2021). Emerging and recurrent plant viruses such as tomato spotted wilt orthortospovirus (TSWV) have seriously affected tobacco production yield and quality. Between November 2018 to April 2019 growing season, large thrips (order Thysanoptera) populations were observed in tobacco fields throughout South Africa's major tobacco-growing areas, and plants were showing severe necrosis, yellowing, stunted growth, ringspot, and leaf curling symptoms, which are usually associated with plant virus infections. The disease incidence was estimated at 30%, as per visual observations. Twenty-two symptomatic leaf tissue samples were collected from tobacco farms in Limpopo, Northwest, and Western cape provinces. Samples were initially screened using the double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) with LOEWE®Fast Kit (LOEWE®, Germany), which detectsTSWV, groundnut ringspot orthotospovirus (GRSV), and tomato chlorotic spot orthotospovirus (TCSV). Nineteen of these samples tested positive for orthortospoviruses. Subsequently, a reverse transcription-polymerase chain reaction was performed on all ELISA-positive samples using orthotospovirus-specific primers gM410 & gM870c targeting the NSm gene (Chen et al., 2012). All samples were positive for orthotospoviruses, as indicated by the presence of a 500bp amplicon. Nineteen purified PCR products were sequenced in the forward and reverse directions using Sanger sequencing at the KwaZulu-Natal Research and Innovation Sequencing Platform (KRISP; South Africa). The sequences were aligned using Muscle in MEGA version X (Kumar et al., 2018) to generate the consensus sequences that were then subjected to the standard nucleotide basic local alignment search tool (blastn) on the NCBI website. Blastn analysis showed that 16 of the 19 samples matched TSWV nucleotide sequences, while three of the sequences; LP14 (accession number OL505552), LP18 (accession number OL505553), and NW4 (accession number OL505554), had a nucleotide sequence identity of 96,38%, 96,97% and 97,29% with the GSRV isolate SA-05 (accession number MH742957; Silva et al., 2019) from South Africa, respectively. This is the first report of GRSV infecting tobacco after TSWV was reported in SA. Previously, GSRV was reported on soybean and groundnut in South Africa by Petersen and Morris in (2002) and Silva et al. in (2019). The results from this study lay a foundation for more detailed studies on GRSV, which will result in the development of effective and sustainable control strategies to manage GRSV diseases on tobacco in SA.

High-Throughput Sequencing Application in the Diagnosis and Discovery of Plant-Infecting Viruses in Africa, A Decade Later.

High-throughput sequencing (HTS) application in the field of plant virology started in 2009 and has proven very successful for virus discovery and detection of viruses already known. Plant virology is still a developing science in most of Africa; the number of HTS-related studies published in the scientific literature has been increasing over the years as a result of successful collaborations. Studies using HTS to identify plant-infecting viruses have been conducted in 20 African countries, of which Kenya, South Africa and Tanzania share the most published papers. At least 29 host plants, including various agricultural economically important crops, ornamentals and medicinal plants, have been used in viromics analyses and have resulted in the detection of previously known viruses and novel ones from almost any host. Knowing that the effectiveness of any management program requires knowledge on the types, distribution, incidence, and genetic of the virus-causing disease, integrating HTS and efficient bioinformatics tools in plant virology research projects conducted in Africa is a matter of the utmost importance towards achieving and maintaining sustainable food security.

Emergence and Full Genome Analysis of Tomato Torrado Virus in South Africa.

Emerging pests and diseases are a major threat to food production worldwide. In a recent survey, Tomato torrado virus (ToTV) was identified on tomato crops in the Limpopo province of South Africa and a first report of the disease was published. In this follow-up study, the full genome sequence of a tomato-infecting isolate of ToTV from South Africa was elucidated. High-throughput sequencing was used to generate the full genome of ToTV infecting tomato crops in South Africa. The longest contig obtained for the RNA-1 and RNA-2 genome of ToTV was comprised of 7420 and 5381 nucleotides (nt), respectively. Blast analysis of the RNA-1 sequence of ToTV from South Africa (ToT-186) matched 99% to a Spanish and Polish isolate; the RNA-2 segment of ToTV from South Africa (ToT-186) matched 99% to ToTV isolates from Italy and Poland, respectively. The information presented in this study will go a long way towards better understanding the emergence and spread of ToTV and devising sustainable management of ToTV diseases.

High-throughput sequencing of virus-infected Cucurbita pepo samples revealed the presence of Zucchini shoestring virus in Zimbabwe.

OBJECTIVES: Plant-infecting viruses remain a serious challenge towards achieving food security worldwide. Cucurbit virus surveys were conducted in Zimbabwe during the 2014 and 2015 growing seasons. Leaf samples displaying virus-like symptoms were collected and stored until analysis. Three baby marrow samples were subjected to next-generation sequencing and the data generated were analysed using genomics technologies. Zucchini shoestring virus (ZSSV), a cucurbit-infecting potyvirus previously described in South Africa was one of the viruses identified. The genomes of the three ZSSV isolates are described analysed in this note. RESULTS: The three ZSSV isolates had the same genome size of 10,297 bp excluding the polyA tail with a 43% GC content. The large open reading frame was found at positions 69 to 10,106 on the genome and encodes a 3345 amino acids long polyprotein which had the same cleavage site sequences as those described on the South African isolate except for the P1-pro site. Genome sequence comparisons of all the ZSSV isolates showed that the isolates F7-Art and S6-Prime had identical sequence across the entire genome while sharing 99.06% and 99.34% polyprotein nucleotide and amino acid sequence identities, respectively with the isolate S7-Prime.

Prevalence, epidemiology and molecular studies of Tomato chlorosis virus (ToCV) in South Africa.

Criniviruses accumulate in the phloem tissue and damage crops by reducing chlorophyll which is essential for plant growth and development. Tomato chlorosis crinivirus (ToCV) is vectored by several whitefly species that damage tomato crops throughout the world. In South Africa, ToCV is a poorly studied pathogen of global economic importance. Therefore, a national survey was initiated to investigate the occurrence and distribution of criniviruses infecting tomato crops in South Africa. Whitefly infested tomato crops exhibiting interveinal leaf chlorosis and chlorotic flecking symptoms were assayed for crinivirus infections using a multiplex reverse transcription polymerase reaction (RT-PCR) approach to assess for the presence of crinivirus species that are known to infect solanaceous hosts. Next-generation sequencing (NGS) was used to generate the complete genome of ToCV from South Africa. Results from the survey indicated that ToCV is presently the only crinivirus species infecting tomatoes in South Africa. Blast analysis showed that the RNA-1 segment of ToCV from South Africa (ToCR1-186) matched 99% to Spanish isolates. On the other hand, the RNA-2 (ToCR2-186) segment matched 98% to a South Korean isolate and three Spanish isolates. Although recombination events were not detected, phylogenetic studies showed inconsistencies in the grouping of RNA-1 and RNA-2 segments for some of the ToCV isolates analyzed in this study. Therefore, we suggest the possibility of intraspecific reassortment. This is the first comprehensive study and full genome sequence of ToCV from South Africa. The information generated from this study is intended to raise awareness of ToCV infections on tomato crops in South Africa.

Genome Sequence of a Tomato-Infecting Tomato Mosaic Virus Isolate from Zimbabwe.

A tomato-infecting tomato mosaic virus (ToMV) isolate was detected in Zimbabwe using lateral flow kits and double-antibody sandwich enzyme-linked immunosorbent assay. Next-generation sequencing and de novo assembly were subsequently performed to determine its genome sequence. The ToMV genome of the Zimbabwe isolate is the second to be reported in Africa.

Pepo aphid-borne yellows virus: a new species in the genus Polerovirus.

Pepo aphid-borne yellows virus (PABYV) has been proposed as a putative representative of a new species in the genus Polerovirus in the family Luteoviridae. The genomes of two South African (SA) isolates of cucurbit-infecting PABYV were described in this record. Total RNA, extracted from a pattypan (Cucurbita pepo L.) and a baby marrow (C. pepo L.) leaf samples, was subjected to next-generation sequencing (NGS) on the HiSeq Illumina platform. Sanger sequencing was subsequently used to authenticate the integrity of PABYV's genome generated from de novo assembly of the NGS data. PABYV genome of SA isolates consists of 5813 nucleotides and displays an organisation typical of poleroviruses. Genome sequence comparisons of the SA PABYV isolates to other poleroviruses support the classification of PABYV as a new species in the genus Polerovirus. Recombination analyses showed that PABYV and Cucurbit aphid-borne yellows virus (CABYV) shared the same ancestor for the genome part situated between breaking points. Phylogenetic analyses of the RNA-dependent RNA polymerase and the coat protein genes showed that SA PABYV isolates shared distant relationship with CABYV and Suakwa aphid-borne yellows virus. Based on our results, we propose that PABYV is a distinct species in the genus Polerovirus.

Genome sequence analysis of two South African isolates of Moroccan watermelon mosaic virus infecting cucurbits.

Moroccan watermelon mosaic virus (MWMV) has been prevalent in cucurbits in the Republic of South Africa (RSA) since it was first reported in 1987. However, full genome studies of the South African isolates have never been conducted previously. The full genome of two MWMV isolates infecting cucurbits (Cucurbita pepo L.) in the province of KwaZulu-Natal, RSA, was compared with the genome of the Tunisian isolate in this communication. The genome sequences of the RSA MWMV isolates were elucidated using next-generation sequencing and Sanger sequencing. The analyses performed included nucleotide and amino acid sequence comparison, determination of the genetic distances, detection of potential recombination, and phylogeny. The genome sequences of the RSA MWMV isolates were found to be 9719 nucleotides long, excluding the poly(A) tail. Sequence homology, genetic distances, and phylogenetic analyses indicated close relationships between the RSA isolates. This record will contribute to building up the MWMV isolate sequences from the different countries where the virus occurs, a useful step toward understanding MWMV evolution.

Review of Beet pseudoyellows virus genome structure built the consensus genome organization of cucumber strains and highlighted the unique feature of strawberry strain.

The complete nucleotide sequences of Beet pseudoyellows virus (BPYV)-MI (cucumber isolate; Matsuyama, Idai) genomic RNAs 1 and 2 were determined and compared with the previously sequenced Japanese cucumber strain (BPYV-JC) and a strawberry strain (BPYV-S). The RNA 2 of BPYV-MI showed 99 % nucleotide sequence identity with both BPYV-JC and -S having highly conserved eight ORFs. In contrast, the RNA1 of BPYV-MI showed sequence identities of 98 and 86 % with BPYV-JC and -S, respectively. Phylogenetic analysis of RNA-dependent RNA polymerase (RdRp) coding sequences from three fully sequenced BPYV strains and five partially sequenced cucurbit-infecting BPYV strains from Japan and South Africa has shown that cucurbit-infecting strains are closer to each other than to BPYV-S. In addition, the strawberry strain BPYV-S has an ORF2 in the downstream of RdRp gene in RNA1, but all the cucumber strains, BPYV-JC, -MI, and those from South Africa, lacked the ORF2 of RNA1, highlighting the difference between common BPYV cucumber strains and a unique strawberry strain.

Full-genome analyses of a Potato Virus Y (PVY) isolate infecting pepper (Capsicum annuum L.) in the Republic of South Africa.

Potato Virus Y (PVY) is a pathogen of economic importance in pepper and other major crop species in the family Solanaceae. Three major PVY strain groups: O, C, and N, have been distinguished on the basis of genome sequencing. In this study, the first full-genome sequence of a PVY isolate (JVW-186) infecting pepper from the province of KwaZulu-Natal, Republic of South Africa is reported. The complete genome sequence of JVW-186 was assembled from overlapping RT-PCR clones using MEGA 5 software. Two ORFs were identified at position 186 and 2915 of the sequence encoding the viral polyprotein and the frameshift translated protein P3N-PIPO, respectively. RDP4 software confirmed three recombination breakpoints at position 343, 1365, and 9308 of the sequence. At each recombination event, a 1,021-bp fragment at the 5' end in the region of the P1/HC-Pro protein and a 392-bp fragment in the region of the coat protein shared a high sequence similarity of 91.8 and 98.89 % to the potato borne PVY(C) isolate PRI-509 and the PVY(O) isolate SASA-110, respectively. The non-recombinant fragment 1 (342-bp) clustered within the C clade of PVY isolates; however, the large 7,942-bp fragment 3 did not cluster within any of the clades. This suggests the possibility of a PVY isolate that has evolved due to the dynamics of selection pressure or the likelihood of an ancestral PVY strain.