Metaviromic Characterization of Betaflexivirus Populations Associated with a Vitis cultivar Collection in South Africa.

South Africa is associated with a centuries-old viticultural industry, accompanied by a diverse range of wine and table grape cultivars and an extensive history of pervasive introductions of vine material and associated viruses. The Vitis D2 collection in Stellenbosch represents the most comprehensive collection of Vitis species, hybrids, and cultivars in South Africa. We collected leaf petiole material from 229 accessions from this collection. Our metaviromic analyses revealed a total of 406 complete/near complete genomes of various betaflexiviruses. Among these, we identified the presence of grapevine rupestris stem pitting-associated virus and grapevine viruses A, B, E, F, H (GVH), I (GVI), and M (GVM). Notably, this study marks the first report of GVH, GVI, and GVM in South Africa, which were confirmed via RT-PCR. This research significantly contributes to our understanding of viral diversity and introductions in South African viticulture and emphasizes the need for vigilant monitoring and management of viral infections. Our findings lay the groundwork for strategies that mitigate the impact of viruses on South Africa's wine industry, which generates an annual revenue of approximately 500 million USD.

Diversity of viroids infecting grapevines in the South African Vitis germplasm collection.

Seven viroid species and one putative viroid species have been reported to infect grapevine namely, hop stunt viroid (HSVd), grapevine yellow speckle viroid 1 (GYSVd-1), grapevine yellow speckle viroid 2 (GYSVd-2), Australian grapevine viroid (AGVd), Japanese grapevine viroid (JGVd), grapevine latent viroid (GLVd), and citrus exocortis viroid (CEVd), as well as a grapevine hammerhead viroid-like RNA (GHVd), so far. In this study, RNA sequence (RNA-Seq) data, from 229 Vitis accessions from the field-maintained vineyard of the South African Vitis germplasm collection, were analysed to determine the diversity of the viroids present. Five of the seven known grapevine-infecting viroids and one putative grapevine-infecting viroid species were very commonly found, with 214 of the 229 samples containing at least one viroid species. HSVd, GYSVd-1, GYSVd-2, AGVd, and JGVd, as well as GHVd, were identified in the RNA-Seq data of the samples and confirmed using RT-PCR and Sanger sequencing. The HSVd sequences indicated the presence of two variants, with one showing multiple nucleotide insertions. AGVd and GYSVd-2 did not display significant sequence diversity, confirming past international studies. GYSVd-1 occurs as four major variants worldwide and representatives of all four variants were identified in this vineyard. This is the first report on the diversity of viroids infecting grapevine in South Africa and the first report of JGVd outside of Japan and GHVd in South Africa. Further studies are needed to fully assess the population and to identify potentially new viroid species.

Genetic diversity of Diaphorina citri (Hemiptera: Liviidae) unravels phylogeographic structure and invasion history of eastern African populations.

The Asian citrus psyllid (Diaphorina citri Kuwayama) is a key pest of Citrus sp. worldwide, as it acts as a vector for Candidatus Liberibacter asiaticus, the bacterial pathogen that causes citrus Huanglongbing. Diaphorina citri has been reported in Kenya, Tanzania, and more recently in Ethiopia. This study assessed the genetic diversity and phylogeographic structure of the pest to gain insights into the potential sources of its introduction into Africa. Population structure and differentiation of D. citri populations from China, Ethiopia, Kenya, Tanzania, and the USA were assessed using 10 microsatellite loci. Additionally, five new complete mitogenomes of D. citri collected in China, Ethiopia, Kenya, Tanzania, and the USA were analyzed in the context of publicly available sequences. Genotype data grouped the D. citri populations from Kenya and Tanzania in one cluster, and those from Ethiopia formed a separate cluster. The two genetic clusters inferred from genotype data were congruent with mitochondrial sequence data. The mitogenomes from Kenya/Tanzania/China had 99.0% similarity, and the Ethiopia/USA had 99.9% similarity. In conclusion, D. citri populations in eastern Africa have different sources, as the Kenyan and Tanzanian populations probably originated from southeastern Asia, while the Ethiopian population most probably originated from the Americas.

Genomic characterization of soybean blotchy mosaic virus, a cytorhabdovirus from South Africa.

Samples showing blotchy mottle symptoms were collected from soybeans in North-West province, South Africa. The assembly of high-throughput sequencing data from three samples yielded contigs of 13,426 to 13,435nt, which represent the first complete genome sequences of soybean blotchy mosaic virus (SbBMV). SbBMV shows a typical cytorhabdovirus gene organization (3'-N-P-P3-M-G-L-5'), with each putative gene product being most similar, but with only 49.1-71.1% sequence identity, to those of cucurbit cytorhabdovirus 1. Given the species demarcation thresholds for rhabdoviruses, SbBMV is thus a distinct member of the genus Cytorhabdovirus.

Genome-Informed Design of a LAMP Assay for the Specific Detection of the Strain of 'Candidatus Phytoplasma asteris' Phytoplasma Occurring in Grapevines in South Africa.

Grapevine yellows is one of the most damaging phytoplasma-associated diseases worldwide. It is linked to several phytoplasma species, which can vary regionally due to phytoplasma and insect-vector diversity. Specific, rapid, and reliable detection of the grapevine yellows pathogen has an important role in phytoplasma control. The purpose of this study was to develop and validate a specific loop-mediated isothermal amplification (LAMP) assay for detection of a distinct strain of grapevine 'Candidatus Phytoplasma asteris' that is present in South Africa, through implementation of a genome-informed test design approach. Several freely available, user-friendly, web-based tools were coupled to design the specific LAMP assays. The criteria for selection of the assays were set for each step of the process, which resulted in four experimentally operative LAMP assays that targeted the ftsH/hflB gene region, specific to the aster yellows phytoplasma strain from South Africa. A real-time PCR was developed, targeting the same genetic region, to provide extensive validation of the LAMP assay. The validated molecular assays are highly specific to the targeted aster yellows phytoplasma strain from South Africa, with good sensitivity and reproducibility. We show a genome-informed molecular test design and an efficient validation approach for molecular tests if reference and sample materials are sparse and hard to obtain.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Genomic characterization of grapevine viruses N and O: novel vitiviruses from South Africa.

A survey was performed on a Vitis cultivar collection in Stellenbosch, South Africa. Metaviromes were generated for each cultivar, using an RNAtag-seq workflow. Analysis of assembled contigs indicated the presence of two putatively novel members of the genus Vitivirus, provisionally named "grapevine virus N" (GVN) and "grapevine virus O" (GVO). Comparisons of amino acid sequences showed that GVN and GVO are most closely related to grapevine virus G and grapevine virus E, respectively. The incidence of these novel viruses within the sampling site was low, with GVO and GVN associated with only five and two cultivars, respectively, of the 229 sampled.

A Review of the 'Candidatus Liberibacter africanus' Citrus Pathosystem in Africa.

It has been nearly 100 years since citrus growers in two distinct regions in the northern provinces of South Africa noticed unusual symptoms in their citrus trees, causing significant crop losses. They had no idea that these symptoms would later become part of an almost global pandemic of a disease called greening or huanglongbing (HLB). The rapid spread of the disease indicated that it might be caused by a transmissible pathogen, but it took >50 years to identify the causative agent as 'Candidatus Liberibacter africanus'. Recently, the disease appeared in more African countries, spreading by both infected planting material and Trioza erytreae. To date, five 'Ca. L. africanus' subspecies have been identified in various rutaceous species, with 'Ca. L. africanus subsp. clausenae' the only subspecies for which a biovar was detected in citrus. Efforts to detect and differentiate HLB-causing Liberibacter species are ongoing, and recent developments are discussed here. This review focuses on aspects of the African form of HLB, including its specific bacterial species and subspecies, its main insect vector, its geographic distribution, and current management strategies.

Novel viruses associated with plants of the family Amaryllidaceae in South Africa.

Nineteen samples from members of the plant genera Agapanthus, Clivia, Hippeastrum, and Scadoxus were collected from gardens in the Gauteng and Western Cape provinces of South Africa. The plants displayed highly variable symptoms of viral disease, including chlorosis, necrosis, streaking, and ringspot. RNAtag-seq was used to characterize the associated viral populations. Plants of the genus Agapanthus were found to be associated with three novel viruses from the families Caulimoviridae, Closteroviridae, and Betaflexiviridae; plants of the genus Clivia were associated with novel members of the families Potyviridae and Betaflexiviridae; and plants of the genus Scadoxus were associated with a novel member of the family Tospoviridae. Nerine latent virus was associated with plants of the genera Agapanthus, Clivia, and Hippeastrum, while hippeastrum mosaic virus was associated exclusively with a Hippeastrum cultivar.

Complete genome sequence of a grapevine Roditis leaf discoloration-associated virus (GRLDaV) variant from South Africa.

High-throughput sequencing (HTS) was used to construct the virome profile of an old grapevine-leafroll-diseased grapevine (Vitis vinifera). De novo assembly of HTS data showed a complex infection, including a virus sequence with similarity to viruses of the genus Badnavirus, family Caulimoviridae. The complete genome sequence of this virus consists of 7090 nucleotides and has four open reading frames (ORFs). Genome organisation and phylogenetic analysis identify this virus as a divergent variant of grapevine Roditis leaf discoloration-associated virus (GRLDaV) with 90% nucleotide sequence identity to isolate w4 (NC_027131). This is the first genome sequence of a South African variant of GRLDaV.

First report of grapevine virus H (GVH) in grapevine in Greece.

Grapevine virus H (GVH) is a member of the genus Vitivirus in the family Betaflexiviridae (subfamily Trivirinae, order Tymovirales) that infects grapevine (Candresse et al., 2018). GVH was first identified in a symptomless grapevine of an unknown cultivar from Portugal in 2018 (Candresse et al. 2018), and since then the virus has been reported only from California (Diaz­Lara et al. 2019). Several vitiviruses have been detected in Greek vineyards (Avgelis and Roubos 2000; Dovas and Katis 2003a; 2003b; Panailidou et al. 2019; Lotos et al. 2020), but no information was available on the presence of GVH. In the fall of 2020, in order to investigate the virome of a commercial vineyard of the cultivar Assyrtiko in northern Greece, a composite sample was made of leaves and petioles from nine vines exhibiting leafroll disease symptoms. Total RNA was extracted from the composite sample according to the protocol of White et al. (2008) and subjected to rRNA depletion, library construction (TruSeq Stranded Total RNA kit), and high-throughput sequencing (HTS) in a NovaSeq6000 platform (Illumina Inc.) at Macrogen (Korea). The resulting ~42 million 101-nt paired-end reads were analyzed in Geneious Prime 2020, and the assembled de novo contigs were subjected to a local BLASTn search, which revealed the presence of 18 grapevine infecting viruses and viroids, among which also a GVH-like contig (GeA-9). GeA-9 was 7,404 nucleotides (nt) long, covering 99.4% of the full virus genome and shared 98.2 % nt identity with a GVH isolate from the USA (MN716768). To confirm the presence of GVH, the nine samples of the cultivar Assyrtiko, used initially to produce the composite sample for HTS analysis, were tested individually by RT-PCR, using the primers GVH_F_2504 (5'-CTGCTTCGCTGAACATATGC-3') and GVH_R_2835 (5'-ATCATTRTGATCGAGAGAGTAGTG-3') that amplify a 331-nt segment of ORF1. GVH was detected in five out of the nine tested samples and one of these was reamplified and subjected to Sanger sequencing. The fragment of ORF1 obtained by Sanger sequencing (MW460005) was 97.5% identical to the nucleotide sequence of the corresponding GVH-like de novo contig (GeA-9) from HTS analysis and it shared 97.2% nt identity with GVH sequences reported from Portugal and USA, respectively (NC_040545 and MN716768), confirming the presence of GVH in the tested samples. This is the first report of GVH in grapevine in Greece, thus further increasing the number of vitiviruses known to infect Greek vineyards and also expanding the number of geographic locations in which GVH is recorded so far.

Author Correction: Mitochondrial genetic variation reveals phylogeographic structure and cryptic diversity in Trioza erytreae.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Mitochondrial genetic variation reveals phylogeographic structure and cryptic diversity in Trioza erytreae.

Trioza erytreae is the main vector for 'Candidatus Liberibacter africanus', the causative agent of African Citrus Greening disease. The insect is widespread in Africa, and has recently disseminated to Southwestern Europe. This study aimed at generating reference mitogenome sequences for T. erytreae, as a background for future genetic diversity surveys. Complete mitochondrial sequences of three specimens collected in Ethiopia, Uganda and South Africa were recovered using Ion Torrent technology. The mitogenomes of T. erytreae from Uganda and Ethiopia were highly similar, and distinct from that found in South Africa. The phylogeographic structure of T. erytreae was assessed using genetic clustering and pairwise distances, based on a dataset of public COI sequences recorded as T. erytreae. The dataset revealed ten haplotypes with strong phylogeographic structure in Africa and Europe. Three haplotypes found in Kenya on Clausena anisata belonged to pairs separated by distances as high as 11.2%, and were basal to all other sequences. These results indicate that not all sequences identified as T. erytreae belong to the same species, and that some degree of specificity with different plant hosts is likely to exist. This study provides new baseline information on the diversity of T. erytreae, with potential implications for the epidemiology of African Citrus Greening disease.

Detection of Asian Citrus Psyllid (Hemiptera: Psyllidae) in Ethiopia: A New Haplotype and its Implication to the Proliferation of Huanglongbing.

Diaphorina citri Kuwayama, also known as the Asian citrus psyllid, is a pest of citrus known for its transmission of Candidatus Liberibacter asiaticus (Ca. L. asiaticus), the causal bacterium of Huanglongbing. The African citrus triozid Trioza erytreae (Del Guercio) (Hemiptera: Triozidae) has been the putative vector of Candidatus Liberibacter africanus (Ca. L. africanus) which causes the African citrus greening disease, until the recent detection of D. citri on the continent. Following reports of D. citri in Kenya and Tanzania, we surveyed citrus plants to establish the presence/absence of D. citri in Ethiopia in citrus-growing regions ranging from 900 to 2,460 m above sea level (masl). Diaphorina citri adults were detected in five of the surveyed sites in Ethiopia. Adult insects encountered were collected using an aspirator and stored in 97% ethanol. The mitochondrial cytochrome oxidase 1 (mt COI) gene of the collected insects was amplified using LepF1/LepR1 primers, and sequences obtained showed low variation, which fell within the acceptable range of species. BLAST was used to query the sequences obtained, and all the sequences linked to D. citri accessions that are available in GenBank. The analysis of the sequences revealed a new haplotype of the species that differs from haplotypes previously reported. Phylogenetic relationships of our samples and other D. citri reference sequences was inferred using the Maximum-likelihood method. Monophyly was observed between the samples and the publicly available sequences from global accessions. This is the first report of the presence of D. citri in Ethiopia.

Microbiome diversity in Diaphorina citri populations from Kenya and Tanzania shows links to China.

The Asian citrus psyllid (Diaphorina citri) is a key pest of Citrus spp. worldwide, as it acts as a vector for "Candidatus Liberibacter asiaticus (Las)", the bacterial pathogen associated with the destructive Huanglongbing (HLB) disease. Recent detection of D. citri in Africa and reports of Las-associated HLB in Ethiopia suggest that the citrus industry on the continent is under imminent threat. Endosymbionts and gut bacteria play key roles in the biology of arthropods, especially with regards to vector-pathogen interactions and resistance to antibiotics. Thus, we aim to profile the bacterial genera and to identify antibiotic resistance genes within the microbiome of different populations worldwide of D. citri. The metagenome of D. citri was sequenced using the Oxford Nanopore full-length 16S metagenomics protocol, and the "What's in my pot" (WIMP) analysis pipeline. Microbial diversity within and between D. citri populations was assessed, and antibiotic resistance genes were identified using the WIMP-ARMA workflow. The most abundant genera were key endosymbionts of D. citri ("Candidatus Carsonella", "Candidatus Profftella", and Wolbachia). The Shannon diversity index showed that D. citri from Tanzania had the highest diversity of bacterial genera (1.92), and D. citri from China had the lowest (1.34). The Bray-Curtis dissimilarity showed that China and Kenya represented the most diverged populations, while the populations from Kenya and Tanzania were the least diverged. The WIMP-ARMA analyses generated 48 CARD genes from 13 bacterial species in each of the populations. Spectinomycin resistance genes were the most frequently found, with an average of 65.98% in all the populations. These findings add to the knowledge on the diversity of the African D. citri populations and the probable introduction source of the psyllid in these African countries.

Distribution of Candidatus Liberibacter species in Eastern Africa, and the First Report of Candidatus Liberibacter asiaticus in Kenya.

Huanglongbing (HLB) is a serious disease of Citrus sp. worldwide. In Africa and the Mascarene Islands, a similar disease is known as African citrus greening (ACG) and is associated with the bacterium Candidatus Liberibacter africanus (Laf). In recent years, Candidatus Liberibacter asiaticus (Las) associated with the severe HLB has been reported in Ethiopia. Thus, we aimed to identify the Liberibacter species affecting citrus, the associated vectors in Eastern Africa and their ecological distribution. We assessed the presence of generic Liberibacter in symptomatic leaf samples by quantitative PCR. Subsequently, we sequenced the 50 S ribosomal protein L10 (rplJ) gene region in samples positive for Liberibacters and identified the species by comparison with public sequence data using phylogenetic reconstruction and genetic distances. We detected generic Liberibacter in 26%, 21% and 66% of plants tested from Uganda, Ethiopia and Kenya, respectively. The rplJ sequences revealed the most prevalent Liberibacters in Uganda and Ethiopia were LafCl (22%) and Las (17%), respectively. We detected Las in Kenya for the first time from three sites in the coastal region. Finally, we modelled the potential habitat suitability of Las in Eastern Africa using MaxEnt. The projection showed large areas of suitability for the pathogen in the three countries surveyed. Moreover, the potential distribution in Eastern Africa covered important citrus-producing parts of Ethiopia, Kenya, Uganda and Tanzania, and included regions where the disease has not been reported. These findings will guide in the development of an integrated pest management strategy to ACG/HLB management in Africa.

Analysis of Genotype Composition of Citrus tristeza virus Populations Using Illumina Miseq Technology.

Recent research describing the strain-specific mechanisms underlying experimental CTV superinfection exclusion has far-reaching implications for the manner in which cross-protecting sources should be selected for. The strain composition of both cross-protecting sources and field populations needs to be sufficiently characterized to improve control of severe stem-pitting and decline isolates. Many of the biological, serological, and molecular techniques used in previous studies yield very limited information about the strain composition of populations and the relative titer of their components. In this chapter we describe a protocol for the characterization of CTV populations, based on the use of the next-generation sequencing Illumina MiSeq platform of p33 gene amplicons.

Draft Genome Sequence of a "Candidatus Phytoplasma asteris"-Related Strain (Aster Yellows, Subgroup 16SrI-B) from South Africa.

Here, we report the draft genome sequence of a phytoplasma discovered in grapevine. The genome size is 600,116 nucleotides (nt), with 597 predicted open reading frames. It is most similar to a maize bushy stunt phytoplasma of group 16SrI-B (aster yellows). The possible presence of a 3,833-nt plasmid was also noted.

Molecular characterization of Morogoro maize-associated virus, a nucleorhabdovirus detected in maize (Zea mays) in Tanzania.

RNAtag-seq of maize samples collected in Tanzania revealed the presence of a previously undescribed nucleorhabdovirus, tentatively named "Morogoro maize-associated virus" (MMaV), in three samples. The MMaV genome is 12,185-12,187 nucleotides long and shares a 69-70% nucleotide sequence identity with taro vein chlorosis virus. Annotation of the genomes showed a typical nucleorhabdovirus gene organization. PCR was unable to detect the same virus in the remaining 35 samples collected in the region.

Diversity and distribution of Maize-associated totivirus strains from Tanzania.

Typically associated with fungal species, members of the viral family Totiviridae have recently been shown to be associated with plants, including important crop species, such as Carica papaya (papaya) and Zea mays (maize). Maize-associated totivirus (MATV) was first described in China and more recently in Ecuador, where it has been found to co-occur with other viruses known to elicit maize lethal necrosis disease (MLND). In a survey for maize-associated viruses, 35 samples were selected for Illumina HiSeq sequencing, from the Tanzanian maize producing regions of Mara, Arusha, Manyara, Kilimanjaro, Morogoro and Pwani. Libraries were prepared using an RNA-tag-seq methodology. Taxonomic classification of the resulting datasets showed that 6 of the 35 samples from the regions of Arusha, Kilimanjaro, Morogoro and Mara, contained reads that were assigned to MATV reference sequences. This was confirmed with PCR and Sanger sequencing. Read assembly of the six MATV-associated datasets yielded partial MATV genomes, two of which were selected for further characterization, using RACE. This yielded two full-length MATV genomes, one of which is divergent from other available MATV genomes.

The structure and function of the global citrus rhizosphere microbiome.

Citrus is a globally important, perennial fruit crop whose rhizosphere microbiome is thought to play an important role in promoting citrus growth and health. Here, we report a comprehensive analysis of the structural and functional composition of the citrus rhizosphere microbiome. We use both amplicon and deep shotgun metagenomic sequencing of bulk soil and rhizosphere samples collected across distinct biogeographical regions from six continents. Predominant taxa include Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes. The core citrus rhizosphere microbiome comprises Pseudomonas, Agrobacterium, Cupriavidus, Bradyrhizobium, Rhizobium, Mesorhizobium, Burkholderia, Cellvibrio, Sphingomonas, Variovorax and Paraburkholderia, some of which are potential plant beneficial microbes. We also identify over-represented microbial functional traits mediating plant-microbe and microbe-microbe interactions, nutrition acquisition and plant growth promotion in citrus rhizosphere. The results provide valuable information to guide microbial isolation and culturing and, potentially, to harness the power of the microbiome to improve plant production and health.